Redox signaling gel formulation

ABSTRACT

Formulations containing reactive oxygen species (ROS), processes for making these formulations, and methods of using these formulations are described. The formulations can include gels or hydrogels that contain at least one reactive oxygen species (ROS). The formulations can include a composition containing a reduced species (RS) and a reactive oxygen species (ROS). The formulations can also contain a rheology modifier and can include gels or hydrogels. Methods of preparing the formulations can include preparing a composition. Compositions can be prepared by providing water, purifying the water to produce ultra-pure water, combining sodium chloride to the ultra-pure water to create salinated water, and electrolyzing the salinated water at a temperature between about 4.5 to about 5.8° C.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNo. 61/895,134 filed Oct. 24, 2013, titled Redox Signaling GelFormulation. The disclosure of each of the applications to which thepresent application claims priority are incorporated by reference.

BACKGROUND

It has long been known that the electrolysis of fluids can result inuseful products. Thus, various apparatus and methods have been proposedfor electrolyzing saline solution, however, all of the previouslyavailable schemes present one or more drawbacks.

For example U.S. Pat. No. 8,323,252 to Alimi et al. teaches a gelformulation for the treatment of diabetic foot ulcer and is incorporatedherein by reference in its entirety. Similarly, U.S. Patent ApplicationNo. 2012/0164235 to Northey teaches a hydrogel comprising oxidativereductive potential water and is incorporated herein by reference in itsentirety.

For example U.S. Pat. No. 7,691,249 teaches a method and apparatus formaking electrolyzed water comprising an insulating end cap for acylindrical electrolysis cell and is incorporated herein by reference inits entirety.

For example, U.S. Pat. Nos. 4,236,992 and 4,316,787 to Themy disclose anelectrode, method and apparatus for electrolyzing dilute salinesolutions to produce effective amounts of disinfecting agents such aschlorine, ozone and hydroxide ions. Both of these references areincorporated herein by reference in their entireties

U.S. Pat. No. 5,674,537, U.S. Pat. No. 6,117,285 and U.S. Pat. No.6,007,686 also teach electrolyzed fluids and are now incorporated hereinby reference in their entireties.

U.S. Pat. No. 4,810,344 teaches a water electrolyzing apparatusincluding a plurality of electrolysis devices, each comprising anelectrolysis vessel having a cathode and an anode oppose to each otherand an electrolysis diaphragm partitioning the space between both of theelectrodes wherein the plurality of devices are connected in a seriessuch that only one of the two ionized water discharge channels of thedevices constitutes a water supply channel to the device a thesucceeding stage and is incorporated herein by reference in itsentirety.

U.S. Pat. No. 7,691,249 is now incorporated herein by reference in itsentirety and is directed to a method and apparatus for makingelectrolyzed water.

Methods for treatment of physiological fluids using electrolyzedsolutions are set forth in U.S. Pat. No. 5,334,383 which is nowincorporated herein by reference in its entirety teaches an electrolyzedsaline solution, properly made and administered in vivo, as effective inthe treatment of various infections brought on by invading antigens andparticularly viral infections.

U.S. Pat. No. 5,507,932 which is now incorporated herein by reference inits entirety teaches an apparatus for electrolyzing fluids.

U.S. Pat. No. 8,062,501 is directed to a method for producing neutralelectrolytic water containing OH, O₂, HD and HOO as active elements andis incorporated herein by reference in its entirety.

There is a need for stabilized or contained superoxides, hydroxylradicals and/or OOH* in an aqueous medium, without solvents orcatalysts, outside the human body. The art teaches that superoxides,hydroxyl radicals and/or OOH* last for a very short amount of time.Stabilizing superoxides in particular has proven difficult. (Hayyan etal. Generation and stability of superoxide ion in tris(pentafluoroethyl)trifluorophosphate anion-based ionic liquids, Journal of FluorineChemistry, Volume 142, October 2012, pages 83-89 and Hayyan et al., Longterm stability of superoxide ion in piperidinium, pyrrolidinium andphosphonium cations-based ionic liquids and its utilization in thedestruction of chlorobenzenes, Journal of Electroanalytical Chemistry,Volume 664, 1 Jan. 2012, pages 26-32.)

At the time the priority document was filed, superoxides were known tohave a very short lifespan. (Kahn et al., SPIN TRAPS: IN VITRO TOXICITYAND STABILITY OF RADICAL ADDUCTS, Free Radical Biology & Medicine, Vol.34, No. 11, pp. 1473-1481, 2003. AlNashef et al., ElectrochemicalGeneration of Superoxide in Room-Temperature Ionic Liquids.Electrochemical and Solid State Letters, 4 (11) 016-018 (2001). AlNashefet al., Superoxide Electrochemistry in an Ionic Liquid. Ind. Eng. Chem.Res. 2002, 41, 4475-4478. Bielski et al., Reactivity of HO₂/O₂ Radicalsin Aqueous Solution, J. Phys. Chem. Ref. Data, Vol. 14, No. 4 1985.Konaka et al., IRRADIATION OF TITANIUM DIOXIDE GENERATES BOTH SINGLETOXYGEN AND SUPEROXIDE ANION, Free Radical Biology & Medicine, Vol. 27,Nos. 3/4, pp. 294-300, 1999.)

As described in the art, the process of making electrolyzed waterrequires membranes. (Zhuang et al., Homogeneous blend membrane made frompoly(ether sulphone) and poly(vinylpyrrolidone) and its application towater electrolysis, Journal of Membrane Science, Volume 300, Issues 1-2,15 Aug. 2007, pages 205-210. Sawada et al., Solid polymer electrolytewater electrolysis systems for hydrogen production based on our newlydeveloped membranes, Part I: Analysis of voltage. Progress in NuclearEnergy, Volume 50, Issues 2-6, March-August 2008, pages 443-448. Okadaet al., Theory for water management in membranes for polymer electrolytefuel cells: Part 1. The effect of impurity ions at the anode side on themembrane performances, Journal of Electroanalytical Chemistry, Volume465, Issue 1, 6 Apr. 1999, pages 1-17. Okada et al. Theory for watermanagement in membranes for polymer electrolyte fuel cells: Part 2. Theeffect of impurity ions at the cathode side on the membraneperformances, Journal of Electroanalytical Chemistry, Volume 465, Issue1, 6 Apr. 1999, pages 18-29. Okada et al., Ion and water transportcharacteristics of Nafion membranes as electrolytes, ElectrochimicaActa, Volume 43, Issue 24, 21 Aug. 1998, pages 3741-3747. Zoulias etal., (2004), A review on water electrolysis, TCJST, 4(2), 41-71. Xu etal., Ion exchange membranes: state of their development and perspective,Journal of Membrane Science, 263 (2005) 1-29. Kariduraganavar et al.,Ion-exchange membranes: preparative methods for electrodialysis and fuelcell applications, Desalination 197 (2006) 225-246. Asawa et al.,Material properties of cation exchange membranes for chloralkalielectrolysis, water electrolysis, and fuel cells, Journal of AppliedElectrochemistry, July 1989, Volume 19, Issue 4, pp 566-570.) Therefore,there is a need for a process to prepare electrolyzed water without aseparator or separating membrane/diaphragm.

Reactive oxygen species (ROS) are important in a variety of fields. Inmedicine there is evidence linking ROS to the aging, disease processes,and the reduction of oxidative stress. Furthermore, ROS are employed asmicrobicidal agents in the home, hospital and other settings. ROS alsoinclude superoxides.

Redox signaling deals with the action of a set of several simplereactive signaling molecules that are mostly produced by mitochondriaresiding inside cells during the metabolism of sugars. These reactivesignaling molecules are categorized into two general groups, ReactiveOxygen Species (ROS), which contain oxidants, and Reduced Species (RS),which contain reductants. These fundamental universal signalingmolecules in the body are the simple but extremely important reactivesignaling molecules that are formed from combinations of the atoms (Na,Cl, H, O, N) that are readily found in the saline bath that fills theinside of the cells (cytosol). All of the molecular mechanisms insidehealthy cells float around in this saline bath and are surrounded by abalanced mixture of such reactive signaling molecules. A few examples ofthe more than 20 reactive molecules formed from these atoms inside thecell, some of which are discussed herein, are superoxide, hydrogenperoxide, hypochlorous acid and nitric oxide.

Such reactive signaling molecules are chemically broken down byspecialized enzymes placed at strategic locations inside the cell. Someof these protective enzymes are classified as antioxidants such asGlutathione Peroxidase and Superoxide Dismutase. In a healthy cell, themixtures of these reactive signaling molecules are broken down by theantioxidant enzymes at the same rate that they are produced by themitochondria. As long as this homeostatic balance is maintained, thecell's chemistry is in balance and all is well.

When damage occurs to the cell, for any number of reasons, includingbacterial or viral invasion, DNA damage, physical damage or toxins, thishomeostatic balance is disturbed and a build-up of oxidants orreductants occurs in the cell. This condition is known as oxidativestress and it acts as a clear signal to the cell that something iswrong. The cell reacts to this signal by producing the enzymes andrepair molecules necessary to attempt repairs to the damage and it alsocan send messengers to activate the immune system to identify andeliminate threats. If oxidative stress persists in the cell for morethan a few hours, then the cell's repair attempts are consideredunsuccessful and the cell kills and dismantles itself and is replaced bythe natural cellular division of healthy neighboring cells.

On a cellular level, this is essentially the healthy tissue maintenanceprocess: damaged cells are detected and repaired or replaced by healthycells. This cellular repair and regeneration process is constantlytaking place, millions of times an hour, in all parts of the body.

There is a need in the art for a safe, effective, economical way ofproducing superoxides and employing them in the medical industries.

BRIEF SUMMARY

Described herein are some embodiments of products containing reactiveoxygen species (ROS), processes for making products which contain ROS,and methods of using these products which contain ROS. In otherembodiments, formulations can include gels or hydrogels that can includeat least one reactive oxygen species (ROS). Described herein generallyare aqueous formulations including at least one stable reactive and/orradical species.

In some embodiments, a formulation containing ROS can comprise acomposition comprising reduced species (RS) and reactive oxygen species(ROS), and a rheology modifier. In other embodiments, the reactiveoxygen species (ROS) can comprise at least one superoxide. In yet otherembodiments, the rheology modifier can comprise a metal silicate gellingagent. In some embodiments, the rheology modifier can comprise SiO₂,MgO, Li₂O, Na₂O, or combinations thereof. In other embodiments, therheology modifier can comprise a cross-linked acrylic acid polymer. Inyet other embodiments, the composition can have a pH between about 6 andabout 9. In some embodiments, the formulation can be administered to auser.

In some embodiments, the formulation containing a ROS can comprise acomposition. In other embodiments, the composition can further comprisesodium present at a concentration of about 1000 to about 1400 ppm, withthe sodium measured by inductively coupled plasma mass spectrometry(ICP-MS). In yet other embodiments, the composition can comprisechloride present at a concentration from about 1200 to about 1600 ppm,with the chloride measured by inductively coupled plasma massspectrometry (ICP-MS). In some embodiments, the composition can comprisechloride present at a concentration from about 0 to about 1 ppm, withthe chloride measured by ³⁵Cl nuclear magnetic resonance (³⁵Cl NMR). Inother embodiments, the composition can comprise hypochlorous acidpresent at a concentration of about 16 to about 24 ppm, with thehypochlorous acid measured by colorimetry. In yet other embodiments, thecomposition can comprise hypochlorous acid present at a concentration ofabout 2300 to about 2700 ppm, with the hypochlorous acid measured by³⁵Cl nuclear magnetic resonance (³⁵Cl NMR). In some embodiments, thecomposition can comprise superoxide radical present at a concentrationof about 94 μM, with the superoxide radical measured by5-(Diisopropoxyphosphoryl)-5-1-pyrroline-N-oxide nuclear magneticresonance (DIPPMPO-NMR). In other embodiments, the composition cancomprise hydroxyl radical present at a concentration of about 241 μM,with the hydroxyl radical measured by DIPPMPO-NMR. In other embodiments,the composition can comprise hydroxyl radical present at a concentrationof about 0 to about 10 ppm, with the hydroxyl radical measured by massspectrometry (MS). In yet other embodiments, the composition cancomprise no hydroxyl radical.

In yet other embodiments, the composition can have a pH between about 6and about 9. In some embodiments, the sodium, chloride, hypochlorousacid, superoxide radical and hydroxyl radical can be measured less thanone year after the composition was made. In some embodiments, theformulation can be administered to a user topically. In otherembodiments, the composition can have an electron paramagnetic resonance(EPR) spectrum as shown in FIG. 13. In yet other embodiments, theformulation can further comprise a rheology modifier. In someembodiments, the rheology modifier can comprise SiO₂, MgO, Li₂O, Na₂O, across-linked acrylic acid polymer, poly(acrylic acid), or combinationsthereof.

In some embodiments, methods of preparing a formulation are disclosed.In other embodiments, a method of preparing a formulation of acomposition can comprise preparing a composition. In yet otherembodiments, a method of preparing a composition can comprise providingwater, purifying the water to produce an ultra-pure water, combiningsodium chloride to the ultra-pure water to create a salinated water, andelectrolyzing the salinated water at a temperature of about 4.5 to about5.8° C. In yet other embodiments, the electrolyzing can be accomplishedwith an anode, a cathode and a power source. In some embodiments, thepower source can comprise a transformer and a rectifier. In otherembodiments, the power source may not comprise a filter capacitor. Inyet other embodiments, the method can include a pulsating voltage suchthat the voltage is about zero at least about 50 times per second. Insome embodiments, the method can further comprise combining thecomposition with a rheology modifier. In other embodiments, the rheologymodifier can further comprise SiO₂, MgO, Li₂O, Na₂O, a cross-linkedacrylic acid polymer, poly(acrylic acid), or combinations thereof.

In some embodiments, the method can further comprise ensuring sodium ispresent at a concentration of about 1000 to about 1400 ppm by measuringsodium by inductively coupled plasma mass spectrometry (ICP-MS). Inother embodiments, the method can further comprise ensuring chloride ispresent at a concentration from about 1200 to about 1600 ppm bymeasuring chloride by inductively coupled plasma mass spectrometry(ICP-MS). In yet other embodiments, the method can further compriseensuring chloride is present at a concentration from about 0 to about 1ppm by measuring chloride by ³⁵Cl nuclear magnetic resonance (³⁵Cl NMR).In some embodiments, the method can further comprise ensuringhypochlorous acid is present at a concentration of about 16 to about 24ppm by measuring hypochlorous acid by colorimetry. In other embodiments,the method can further comprise ensuring hypochlorous acid is present ata concentration of about 2300 to about 2700 ppm by measuringhypochlorous acid by ³⁵Cl nuclear magnetic resonance (³⁵Cl NMR). In yetother embodiments, the method can further comprise ensuring superoxideradical is present at a concentration of about 94 μM by measuringsuperoxide radical by 5-(Diisopropoxyphosphoryl)-5-1-pyrroline-N-oxidenuclear magnetic resonance (DIPPMPO-NMR). In some embodiments, themethod can further comprise ensuring hydroxyl radical is present at aconcentration of about 241 μM, by measuring hydroxyl radical byDIPPMPO-NMR. In other embodiments, the method can comprise ensuringhydroxyl radical is present at a concentration of about 0 to about 10ppm by measuring hydroxyl radical by mass spectrometry (MS). In yetother embodiments, the method can further comprise administering theformulation to a user topically.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to describe the manner in which the above-recited and otheradvantages and features of the invention can be obtained, a moreparticular description of the invention briefly described above will berendered by reference to specific embodiments thereof which areillustrated in the appended drawings. Understanding that these drawingsdepict only typical embodiments of the invention and are not thereforeto be considered to be limiting of its scope, the invention will bedescribed and explained with additional specificity and detail throughthe use of the accompanying drawings in which:

FIG. 1 illustrates a flow chart of a process as described herein;

FIG. 2 illustrates an example diagram of the generation of variousmolecules at the electrodes. The molecules written between theelectrodes depict the initial reactants and those on the outside of theelectrodes depict the molecules/ions produced at the electrodes andtheir electrode potentials;

FIG. 3 illustrates a plan view of a process and system for producing acomposition according to the present description;

FIG. 4 illustrates an example system for preparing water for furtherprocessing into a composition described herein;

FIG. 5 illustrates a ³⁵Cl spectrum of NaCl, NaClO solution at a pH of12.48, and a composition described herein (the composition is labeled“ASEA”);

FIG. 6 illustrates a 1H NMR spectrum of a composition of the presentdisclosure;

FIG. 7 illustrates a 31P NMR spectrum of DIPPMPO combined with acomposition described herein;

FIG. 8 illustrates a positive ion mode mass spectrum showing a parentpeak and fragmentation pattern for DIPPMPO with m/z peaks at 264, 222,and 180;

FIG. 9 illustrates oxygen/nitrogen ratios for a composition describedherein compared to water and NaClO (the composition is labeled “ASEA”);

FIG. 10 illustrates chlorine/nitrogen ratios for a composition describedherein compared to water and NaClO (the composition is labeled “ASEA”);

FIG. 11 illustrates ozone/nitrogen ratios for a composition describedherein compared to water and NaClO (the composition is labeled “ASEA”);

FIG. 12 illustrates the carbon dioxide to nitrogen ratio of acomposition as described herein compared to water and NaClO (thecomposition is labeled “ASEA”);

FIG. 13 illustrates an EPR splitting pattern of DIPPMOP/ASEA mixture(the composition in a certain embodiment is “ASEA”);

FIG. 14 illustrates a perspective view of a first presently preferredembodiment of an apparatus for making a product;

FIG. 15 illustrates a detailed top view of the electrode assemblyrepresented in FIG. 14;

FIG. 15A illustrates a side cross sectional view of the electrodeassembly represented in FIG. 15 taken along line 3-3 in FIG. 15;

FIG. 16 shows a block diagram of a second presently preferred embodimentof an apparatus for making a product;

FIG. 17 shows a top view of an electrode assembly preferred for use inthe apparatus represented in FIG. 16;

FIG. 18 shows a cross sectional view taken along line 6-6 of FIG. 17;

FIG. 19 illustrates a block diagram of a power source;

FIG. 20 illustrates a block diagram of another power source;

FIG. 21 illustrates a chart of the relative fluorescence of variouscompositions;

FIG. 22 illustrates a graph of the decay rate of superoxide over aperiod of 1 year;

FIG. 23 shows a graph showing the comparison of the decay rates ofsuperoxide when the mixture is stored in a bottle and when the mixtureis stored in a pouch;

FIG. 24 shows a graph of the Expt. 5f07 ROS Assay;

FIG. 25 shows a graph of an Intraassay Variation Using Two Levels ofMPH;

FIG. 26 illustrates a JEOL DART low temperature sample injection TOFMass Spectrum of a composition showing water clusters [(H₂O)_(n)+H]+with peaks at 37 and 55;

FIG. 27 illustrates a JEOL DART low temperature sample injection TOFMass Spectrum of a composition; and

FIG. 28 illustrates a JEOL DART low temperature sample injection TOFMass Spectrum of a composition showing negative ions peaks at 35 and 37.

DETAILED DESCRIPTION OF THE INVENTION

Described herein are embodiments of products containing reactive oxygenspecies (ROS), processes for making products which contain ROS, andmethods of using these products which contain ROS. In some embodiments,the product containing ROS comprises gel formulations. In otherembodiments, gel formulations include hydrogels, creams, ointments,emollients, balms, liniments, unguents, colloids, emulsions,dispersions, sols, sol-gels, salves, or the like, or combinationsthereof. In some embodiments these gel formulations can be used in thepersonal care or cosmetics industry.

Described herein are embodiments of formulations including gels orhydrogels that can generally include at least one reactive oxygenspecies (ROS). ROS can include, but are not limited to superoxides(O₂*—, HO₂*), hypochlorites (Off, HOCl, NaClO), hypochlorates (HClO₂,ClO₂, HClO₃, HClO₄), oxygen derivatives (O₂, O₃, O₄*—, 1O), hydrogenderivatives (H₂, H⁻), hydrogen peroxide (H₂O₂), hydroxyl free radical(OH*−), ionic compounds (Na⁺, Cl⁻, H⁺, OH⁻, NaCl, HCl, NaOH), chlorine(Cl₂), water clusters (n*H₂O-induced dipolar layers around ions), andcombinations thereof. Some ROS can be electron acceptors and some can beelectron donors.

In some embodiments, gels and hydrogels can be made from aqueousingredients and rheology modifiers. Rheology modifiers can includeNewtonian fluids and ‘soft solids’ (solids which under certainconditions respond with plastic flow rather than by deformingelastically in response to an applied force). Rheology modifiers areused in the cosmetic industry to affect the look and feel of cosmeticsand other products and also to impart other beneficial properties tothese cosmetics. In some embodiments, rheology modifiers can be selectedand used in the formulations of gels based on the desiredcharacteristics of the rheology modifier and on the compatibility of therheology modifier with redox signaling compositions.

In some embodiments, rheology modifiers, also called thickening agents,viscosity modifiers or gelling agents, can include acrylic acid-basedpolymers. Acrylic acid-base polymers can include high molecular weight,cross-linked, acrylic acid-based polymers, such as poly(acrylic acid),PAA, carbomer, or polymers having the general structure of (C₃H₄O₂)_(n).

In some embodiments these polymers are sold under the trade nameCarbopol®. Carbopol® polymers can be supplied as rheology modifiers foruse as thickeners, suspending agents, and stabilizers in a variety ofpersonal care products, pharmaceuticals, and household cleaners.Carbopol® polymers may be used in either solid (e.g., powder) or liquidform.

In some embodiments, the acrylic acid-based polymers comprisehomopolymers or copolymers. In other embodiments, suitable homopolymersmay be cross-linked, preferably with allyl sucrose orallylpentaerythritol. In yet other embodiments, suitable copolymers ofacrylic acid can be modified by long chain (C₁₀-C₃₀) alkyl acrylates andcan be cross-linked, e.g., with allylpentaerythritol.

In some embodiments, Carbopol® polymers can be neutralized to achievemaximum viscosity. As supplied, Carbopol® polymers can exist as dry,tightly coiled acidic molecules held in a coiled structure by hydrogenbonds. Once dispersed in water, or another solvent, such polymers canbegin to hydrate and partially uncoil. In other embodiments, Carbopol®polymers may be thickened by converting the acidic polymer to a salt.This can be done by neutralizing with a common base such as sodiumhydroxide (NaOH) or triethanolamine (TEA) to “uncoil” the long chainpolymer and to thicken the polymer. In yet other embodiments, additionalneutralizers can include sodium hydroxide, ammonium hydroxide, potassiumhydroxide, arginine, aminomethyl propanol, tetrahydroxypropyl,ethylenediamine, triethanolamine, trimethamine, PEG-15 Cocamine,diisopropanolamine, and/or triisopropanolamine.

In some embodiments, the amount of neutralizing agent can depend on thedesired characteristics of the gel/hydrogel product and can depend onthe type of neutralizing agent utilized. In other embodiments, theamount of neutralizing agent can be described as a ratio of neutralizerto Carbopol®. In yet other embodiments, the ratio can range from about0.1:1 to 10:1. In some embodiments, the neutralizer can be present in anamount of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3,1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8,2.9, 3, 3.1, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1,5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6,6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1,8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6,9.7, 9.8, 9.9, or 10 parts neutralizer to 1 part Carbopol®. In someembodiments, the neutralizing agent can be NaOH and can be present in aratio of 2.3:1 (neutralizer:Carbopol®). These amounts can be approximateand can be modified to achieve specific characteristics desired and/orrequired in the composition of the formulation.

In some embodiments, suitable thickening agents can yield the desiredviscosity for the formulation, as well as other characteristics, such asappearance, shear resistance, ion resistance, and thermal stability. Inother embodiments, Carbopol® 934 can be used for a formulation that canbe either a suspension or emulsion (rather than a clear gel) with aviscosity greater than 3000 centipoise (cps). In yet other embodiments,Carbopol® 974P may be used for its advantageous bioadhesive properties.In some embodiments, the formulation may comprise Carbopol® Ultrez 30.

In some embodiments, rheology modifiers can include any suitable metalsilicate gelling agent. In other embodiments, a metal silicate gellingagent can be used. In yet other embodiments, a metal silicate with ametal that is an alkali metal, an alkaline earth metal, or anycombinations thereof can be used. In some embodiments, suitable alkalimetals or alkaline earth metals can include, but are not limited to,lithium, sodium, potassium, magnesium, calcium, and the like. In someembodiments, the metal silicate gelling agent can be a sodium magnesiumsilicate or a derivative thereof. In other embodiments, the metalsilicate gelling agent can include sodium magnesium fluorosilicate.

In some embodiments, the rheology modifiers can be present in thehydrogel formulation in any suitable amount. In other embodiments, theformulation comprises about 0.1% by weight to about 10% by weight ofrheology modifier. In yet other embodiments, the amount of modifier canbe from about 1.0% to about 5% by weight. In some embodiments, theamount of modifier can be 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%,0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%,2%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3%, 3.1%,3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4%, 4.1%, 4.2%, 4.3%,4.4%, 4.5%, 4.6%, 4.7%, 4.8%, 4.9%, or 5% by weight. In otherembodiments, the amount of modifier is 1% or 2% by weight. In yet otherembodiments, these weight percentages can be approximate and can bemodified to achieve specific characteristics desired and/or required inthe composition.

In some embodiments, the formulation can include a buffering agent. Inother embodiments, any suitable buffering agent may be employed to yieldand maintain the desired pH of the formulation. In yet otherembodiments, other buffers suitable for use in the hydrogel formulationsdescribed herein can include, but are not limited to, salts and acids ofacetate, glutamate, citrate, tartrate, benzoate, lactate, histidine orother amino acids, gluconate, phosphate, malate, succinate, formate,propionate, and carbonate. In some embodiments, other buffering agentscan be used as generally known in the art (see Handbook of Cosmetic andPersonal Care Additives, 2nd ed., Ashe et al. eds. (2002) and Handbookof Pharmaceutical Excipients, 4th ed., Rowe et al. eds. (2003)). In someembodiments, suitable buffering agents may be either in liquid or solidform. In another embodiment, the buffering agent can be an acid or saltof a phosphate compound. In some embodiments, the buffering agent can besodium phosphate. The sodium phosphate employed herein can be anysuitable form of sodium phosphate including, for example, monobasicsodium phosphate, dibasic sodium phosphate, tetrasodium pyrophosphate,or combinations thereof.

In some embodiments, any suitable amount of buffering agent may beincluded in the formulation. In some embodiments, the amount ofbuffering agent present in the hydrogel formulations can be from about0.01 weight-percent to about 5.0 weight-percent, based on the weight ofthe formulation. In some embodiments, the buffering agent can be presentin an amount of from about 0.1 weight-percent to about 1.0weight-percent.

In some embodiments, the hydrogel formulations may further containadditional components such as colorants, fragrances, buffers,physiologically acceptable carriers and/or excipients, and the like. Insome embodiments, suitable colorants include may, but are not limitedto, titanium dioxide, iron oxides, carbazole violet,chromium-cobalt-aluminum oxide, 4-Bis[(2-hydroxyethyl)amino]-9,10-anthracenedione bis(2-propenoic)ester copolymers, and the like. Insome embodiments, any suitable fragrance can be used.

In some embodiments, the pH of the hydrogel formulation can be generallyfrom about 3 to about 9. In some embodiments, the pH of the hydrogelformulation can be from 5.0 to 7.0. In some embodiments, the pH of thehydrogel formulation can be from 5.6 to 7.0.

In some embodiments, the viscosity of the hydrogel formulation can beany suitable viscosity such that the formulation can be topicallyapplied to a subject. In some embodiments, the viscosity of the hydrogelformulation can be in the range of about 1,000 to about 100,000centipoise (cP). In some embodiments, the viscosity of the hydrogel canbe 1,000 cP, 2,000 cP, 3,000 cP, 4,000 cP, 5,000 cP, 10,000 cP, 15,000cP, 20,000 cP, 25,000 cP, 30,000 cP, 35,000 cP, 40,000 cP, 45,000 cP,50,000 cP, 55,000 cP, 60,000 cP, 65,000 cP, 70,000 cP, 75,000 cP, 80,000cP, 85,000 cP, 90,000 cP, or 95,000 cP. In some embodiments, theviscosity of the hydrogel can be in the range of about 1,000 cP to about20,000 cP. In other embodiments, the viscosity of the hydrogel can be inthe range of about 12,000 cP to about 20,000 cP. These viscosity rangescan be approximate and can be modified to achieve specificcharacteristics desired and/or required in the composition.

In some embodiments, the redox signaling composition can be produced asdescribed herein. Methods of producing these disclosed compositions caninclude one or more of the steps of (1) preparation of an ultra-puresolution of sodium chloride in water, (2) temperature control and flowregulation through a set of inert catalytic electrodes and (3) amodulated electrolytic process that results in the formation of suchstable molecular moieties and complexes: the RS and ROS. In someembodiments, such a process can include the above steps.

In some embodiments, a method of making redox signaling compositionscomprises electrolyzing salinated water having a salt concentration ofabout 2.8 g NaCl/L, using a set of electrodes with an amperage of about3 amps, to form a composition, wherein the water is at or below roomtemperature during 3 minutes of electrolyzing.

In other embodiments, a method of making redox signaling compositionscomprises electrolyzing salinated water having a salt concentration ofabout 9.1 g NaCl/L, using a set of electrodes with an amperage of about3 amps, to form a composition, wherein the water is at or below roomtemperature during 3 minutes of electrolyzing.

In yet other embodiments, the weight percentage of the redox signalingcomposition in the gel can be from about 50 wt % to 99.9 wt %. In someembodiments, the weight percentage of the redox signaling compositioncan be present from 90 to 99.1% by weight or from 95 to 99.1%. In otherembodiments, the amount of redox signaling composition can be present at95.1%, 95.2%, 95.3%, 95.4%, 95.5%, 95.6%, 95.7%, 95.8%, 95.9%, 96.0%,96.1%, 96.2%, 96.3%, 96.4%, 96.5, 96.7, 96.8, 96.9, 97.0, 97.1, 97.2,97.3, 97.4, 97.5, 97.6, 97.7, 97.8, 97.9, 98.0, 98.1, 98.2, 98.3, 98.4,98.5, 98.6, 98.7, 98.8, 98.9, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%,99.6%, 99.7%, 99.8%, or 99.9%. In yet other embodiments, the amount ofthe redox signaling composition can be 98% or 99% by weight. Theseweight percentages can be approximate and can be modified to achievespecific characteristics desired and/or required in the composition.

In some embodiments, water can be supplied from a variety of sources,including but not limited to municipal water, filtered water, nanopurewater, or the like. In other embodiments, as shown in FIG. 1, anoptional reverse osmosis procedure 102 can be used.

In some embodiments, contaminants can be removed from a commercialsource of water by the following procedure: water flows through anactivated carbon filter to remove the aromatic and volatile contaminantsand then undergoes Reverse Osmosis (RO) filtration to remove dissolvedsolids and most organic and inorganic contaminants. The resultingfiltered RO water can contain less than about 8 ppm of dissolved solids.Most of the remaining contaminants can be removed through a distillationprocess, resulting in dissolved solid measurements less than 1 ppm. Inaddition to removing contaminants, distillation may also serve tocondition the water with the correct structure and Oxidation ReductionPotential (ORP) to facilitate the oxidative and reductive reactionpotentials on the platinum electrodes in the subsequentelectro-catalytic process.

In some embodiments, ultra-pure can refer to water which has a totaldissolved solids count of less than 10 ppm. The total dissolved solidscount of less than 10 ppm can be a result of reverse osmosis and/ordistillation. Other known processes for water purification can also beused to reduce the amount of total dissolved solids.

In other embodiments, the reverse osmosis process can vary, but canprovide water having a total dissolved solids content of less than about10 ppm, about 9 ppm, about 8 ppm, about 7 ppm, about 6 ppm, about 5 ppm,about 4 ppm, about 3 ppm, about 2 ppm, about 1 ppm, or the like.

In some embodiments, the reverse osmosis process can be performed at atemperature of about 5° C., about 10° C., about 15° C., about 20° C.,about 25° C., about 30° C., about 35° C., or the like. The reverseosmosis step can be repeated as needed to achieve a particular totaldissolved solids level. In other embodiments, an optional distillationstep 104 can be performed.

In other embodiments, means of reducing contaminants can includefiltration and/or purification such as by utilizing deionization, carbonfiltration, double-distillation, electrodeionization, resin filtrationsuch as with Milli-Q purification, microfiltration, ultrafiltration,ultraviolet oxidation, electrodialysis, or combinations thereof.

In some embodiments, the distillation process can vary, but can providewater having a total dissolved solids content of less than about 5 ppm,about 4 ppm, about 3 ppm, about 2 ppm, about 1 ppm, about 0.9 ppm, about0.8 ppm, about 0.7 ppm, about 0.6 ppm, about 0.5 ppm, about 0.4 ppm,about 0.3 ppm, about 0.2 ppm, about 0.1 ppm, or the like. In otherembodiments the temperature of the distillation process can be performedat a temperature of about 5° C., about 10° C., about 15° C., about 20°C., about 25° C., about 30° C., about 35° C., or the like.

In some embodiments, the distillation step can be repeated as needed toachieve a particular total dissolved solids level. After water has beensubjected to reverse osmosis, distillation, both, or neither, the levelof total dissolved solids in the water can be less than about 5 ppm,about 4 ppm, about 3 ppm, about 2 ppm, about 1 ppm, about 0.9 ppm, about0.8 ppm, about 0.7 ppm, about 0.6 ppm, about 0.5 ppm, about 0.4 ppm,about 0.3 ppm, about 0.2 ppm, about 0.1 ppm, or the like.

In some embodiments, the reverse osmosis step, the distillation step,both, or neither, can be preceded by a carbon filtration step. In otherembodiments, purified water can be used directly with the systems andmethods described herein.

In some embodiments, after water has been subjected to reverse osmosis,distillation, both or neither, or any other purification step asdescribed herein, a salt can be added to the water in a salting step 106of FIG. 1. The salt can be unrefined, refined, caked, de-caked, or thelike. In one embodiment, the salt is sodium chloride (NaCl). In someembodiments, the salt can include an additive. Salt additives caninclude, but are not limited to potassium iodide, sodium iodide, sodiumiodate, dextrose, sodium fluoride, sodium ferrocyanide, tricalciumphosphate, calcium carbonate, magnesium carbonate, fatty acids,magnesium oxide, silicon dioxide, calcium silicate, sodiumaluminosilicate, calcium aluminosilicate, ferrous fumarate, iron, orfolic acid. In some embodiments, additives can be added at this point orat any point during the described process. In other embodiments, theabove additives can be added just prior to bottling.

The saline generally should be free from contaminants, both organic andinorganic, and homogeneous down to the molecular level. In particular,metal ions can interfere with the electro-catalytic surface reactions,and thus it may be helpful for metals to be avoided. In one embodiment,a brine solution is used to salinate the water. The brine solution canhave a NaCl concentration of about 540 g NaCl/gal, such as 537.5 gNaCl/gal.

In another embodiment, the process can be applied to an ionic solublesalt mixture, especially with those containing chlorides. In addition toNaCl, other non-limiting examples include LiCl, HCl, CuCl₂, CuSO₄, KCl,MgCl₂, CaCl₂, sulfates and phosphates. For example, strong acids such assulfuric acid (H₂SO₄), and strong bases such as potassium hydroxide(KOH), and sodium hydroxide (NaOH) are frequently used as electrolytesdue to their strong conducting abilities. Preferably the salt is sodiumchloride (NaCl). A brine solution can be used to introduce the salt intothe water. The amount of brine or salt needs will be apparent to one ofordinary skill in the art.

Salt can be added to water in the form of a brine solution. To mix thebrine solution, a physical mixing apparatus can be used or a circulationor recirculation can be used. In one embodiment, pure pharmaceuticalgrade sodium chloride can be dissolved in the prepared distilled waterto form a 15 wt % sub-saturated brine solution and continuouslyre-circulated and filtered until the salt has completely dissolved andall particles >0.1 microns are removed. This step can take several days.The filtered, dissolved brine solution can then be injected into tanksof distilled water in about a 1:352 ratio (saltwater) in order to form a0.3% saline solution. In one embodiment, a ratio 10.75 g of salt per 1gallon of water can be used to form the composition. In anotherembodiment, 10.75 g of salt in about 3-4 g of water, such as 3,787.5 gof water can be used to form the composition. This solution then can beallowed to re-circulate and diffuse until homogeneity at the molecularscale has been achieved. The brine solution can have a NaClconcentration of about 540 g NaCl/gal, such as 537.5 g NaCl/gal.

Brine can then be added to the previously treated water or to freshuntreated water to achieve a NaCl concentration of between about 1 gNaCl/gal water and about 25 g NaCl/gal water, between about 8 g NaCl/galwater and about 12 g NaCl/gal water, or between about 4 g NaCl/gal waterand about 16 g NaCl/gal water. In a preferred example, the achieved NaClconcentration is 2.8 g/L of water. In another preferred example, theachieved NaCl concentration is 9.1 g/L of water. Once brine is added towater at an appropriate amount, the solution can be thoroughly mixed.The temperature of the liquid during mixing can be at room temperatureor controlled to a desired temperature or temperature range.

To mix the solution, a physical mixing apparatus can be used or acirculation or recirculation can be used. The salt solution can then bechilled in a chilling step 108 of FIG. 1.

For large amounts of composition, various chilling and cooling methodscan be employed. For example cryogenic cooling using liquid nitrogencooling lines can be used. Likewise, the solution can be run throughpropylene glycol heat exchangers to achieve the desired temperature. Thechilling time can vary depending on the amount of liquid, the startingtemperature and the desired chilled temperature.

Products from the anodic reactions can be effectively transported to thecathode to provide the reactants necessary to form the stable complexeson the cathode surfaces. Maintaining a high degree of homogeneity in thefluids circulated between the catalytic surfaces can also be helpful. Aconstant flow of about 2-8 ml/cm² per sec can be used, with typical meshelectrode distances 2 cm apart in large tanks. This flow can bemaintained, in part, by the convective flow of gasses released from theelectrodes during electrolysis.

The mixed solution can then undergo electrochemical processing throughthe use of at least one electrode in an electrolyzing step 110 ofFIG. 1. Each electrode can comprise a conductive metal. Metals caninclude, but are not limited to copper, aluminum, titanium, rhodium,platinum, silver, gold, iron, a combination thereof or an alloy such assteel or brass. The electrode can be coated or plated with a differentmetal such as, but not limited to aluminum, gold, platinum or silver. Inan embodiment, each electrode is formed of titanium and plated withplatinum. The platinum surfaces on the electrodes by themselves can beoptimal to catalyze the required reactions. Rough, double layeredplatinum plating can assure that local “reaction centers” (sharplypointed extrusions) are active and that the reactants not make contactwith the underlying electrode titanium substrate.

In one embodiment, rough platinum-plated mesh electrodes in a vertical,coaxial, cylindrical geometry can be optimal, with, for example, notmore than 2.5 cm, not more than 5 cm, not more than 10 cm, not more than20 cm, or not more than 50 cm separation between the anode and cathode.The amperage run through each electrode can be between about 2 amps andabout 15 amps, between about 4 amps and about 14 amps, at least about 2amps, at least about 4 amps, at least about 6 amps, or any range createdusing any of these values. In one embodiment, 7 amps is used with eachelectrode. In one example, 1 amp is run through the electrodes. In oneexample, 2 amps are run through the electrodes. In one example, 3 ampsare run through the electrodes. In one example, 4 amps are run throughthe electrodes. In one example, 5 amps are run through the electrodes.In one example, 6 amps are run through the electrodes. In one example, 7amps are run through the electrodes. In a preferred example, 3 amps arerun through the electrodes.

The amperage can be running through the electrodes for a sufficient timeto electrolyze the saline solution. The solution can be chilled duringthe electrochemical process. The solution can also be mixed during theelectrochemical process. This mixing can be performed to ensuresubstantially complete electrolysis.

Electric fields between the electrodes can cause movement of ions.Negative ions can move toward the anode and positive ions toward thecathode. This can enable exchange of reactants and products between theelectrodes. In some embodiments, no barriers are needed between theelectrodes.

After amperage has been run through the solution for a sufficient time,an electrolyzed solution is created. The solution can be stored and ortested for particular properties in storage/testing step 112 of FIG. 1.In one embodiment, the homogenous saline solution is chilled to about4.8±0.5° C. Temperature regulation during the entire electro-catalyticprocess is typically required as thermal energy generated from theelectrolysis process itself may cause heating. In one embodiment,process temperatures at the electrodes can be constantly cooled andmaintained at about 4.8° C. throughout electrolysis.

After amperage has been run through the solution for a sufficient time,an electrolyzed solution is created with beneficial properties, such asantifungal properties. The solution can have a pH of about 7.4. In someembodiments, the pH is greater than 7.3. In some embodiments, the pH isnot acidic. In other embodiments, the solution can have a pH less thanabout 7.5. The pH may not be basic. The solution can be stored and ortested for particular properties in a storage/testing step 112 of FIG.1.

The end products of this electrolytic process can react within thesaline solution to produce many different chemical entities. Thecompositions and composition described herein can include one or more ofthese chemical entities, known as redox signaling agents or RXNs.

The chlorine concentration of the electrolyzed solution can be betweenabout 5 ppm and about 34 ppm, between about 10 ppm and about 34 ppm, orbetween about 15 ppm and about 34 ppm. In one embodiment, the chlorineconcentration is about 32 ppm.

The saline concentration in the electrolyzed solution can be, forexample, between about 0.10% w/v and about 0.20% w/v, between about0.11% w/v and about 0.19% w/v, between about 0.12% w/v and about 0.18%w/v, between about 0.13% w/v and about 0.17% w/v, or between about 0.14%w/v and about 0.16% w/v.

The composition can then be bottled in a bottling step 114 of FIG. 1.The composition can be bottled in plastic bottles having volumes ofabout 4 oz, about 8 oz, about 16 oz, about 32 oz, about 48 oz, about 64oz, about 80 oz, about 96 oz, about 112 oz, about 128 oz, about 144 oz,about 160 oz, or any range created using any of these values. Theplastic bottles can also be plastic squeezable pouches having similarvolumes. In one embodiment, plastic squeezable pouches can have one wayvalves to prevent leakage of the composition, for example, duringathletic activity.

During bottling, solution from an approved batch can be pumped through a10 micron filter (e.g., polypropylene) to remove any larger particlesfrom tanks, dust, hair, etc. that might have found their way into thebatch. In other embodiments, this filter need not be used. Then, thesolution can be pumped into the bottles, the overflow going back intothe batch.

Bottles generally may not contain any dyes, metal specks or chemicalsthat can be dissolved by acids or oxidating agents. The bottles, caps,bottling filters, valves, lines and heads used can be specifically berated for acids and oxidating agents. Caps and with organic glues, sealsor other components sensitive to oxidation may be avoided, a these couldneutralize and weaken the product over time.

The bottles and pouches used herein can aid in preventing decay of freeradical species found within the compositions. In other embodiments, thebottles and pouches described do not further the decay process. In otherwords, the bottles and pouches used can be inert with respect to theradical species in the compositions. In one embodiment, a container(e.g., bottle and/or pouch) can allow less than about 10% decay/month,less than about 9% decay/month, less than about 8% decay/month, lessthan about 7% decay/month, less than about 6% decay/month, less thanabout 5% decay/month, less than about 4% decay/month, less than about 3%decay/month, less than about 2% decay/month, less than about 1%decay/month, between about 10% decay/month and about 1% decay/month,between about 5% decay/month and about 1% decay/month, about 10%decay/month, about 9% decay/month, about 8% decay/month, about 7%decay/month, about 6% decay/month, about 5% decay/month, about 4%decay/month, about 3% decay/month, about 2% decay/month, or about 1%decay/month of free radicals in the composition. In one embodiment, abottle can only result in about 3% decay/month of superoxide. In anotherembodiment, a pouch can only result in about 4% decay/month ofsuperoxide.

A direct current, DC, power source can be used to electrolyze water.

The variables of voltage, amps, frequency, time and current requireddepend on the compound and/or ion themselves and their respective bondstrengths. To that end, the variables of voltage, amps, frequency, timeand current are compound and/or ion dependent and are not limitingfactors. That notwithstanding, the voltage used can be less than 40V,such as 30V or 20V or 10V or any voltage in between. The voltage canalso modulate and at any time vary within a range of from 1 to 40V orfrom 10 to 30V or from 20 to 30V. In one embodiment, the voltage canrange during a single cycle of electrolyzing. The range can be from 1 to40V or from 10 to 30V or from 20 to 30V. These ranges are non-limitingbut are shown as examples.

Waveforms with an AC ripple also referred to as pulse or spikingwaveforms include: any positive pulsing currents such as pulsed waves,pulse train, square wave, sawtooth wave, spiked waveforms, pulse-widthmodulation (PWM), pulse duration modulation (PDM), single phase halfwave rectified AC, single phase full wave rectified AC or three phasefull wave rectified for example.

A bridge rectifier may be used. Other types of rectifiers can be usedsuch as Single-phase rectifiers, Full-wave rectifiers, Three-phaserectifiers, Twelve-pulse bridge, Voltage-multiplying rectifiers, filterrectifier, a silicon rectifier, an SCR type rectifier, a high-frequency(RF) rectifier, an inverter digital-controller rectifier, vacuum tubediodes, mercury-arc valves, solid-state diodes, silicon-controlledrectifiers and the like. Pulsed waveforms can be made with a transistorregulated power supply, a dropper type power supply, a switching powersupply and the like.

A transformer may be used. Examples of transformers that can be usedinclude center tapped transformers, autotransformers, capacitor voltagetransformers, distribution transformers, power transformers, phase angleregulating transformers, Scott-T transformers, polyphase transformers,grounding transformers, leakage transformers, resonant transformers,audio transformers, output transformers, laminated core toroidalautotransformers, variable autotransformers, induction regulators, strayfield transformers, solyphase transformer, constant voltage transformer,ferrite core planar transformers, oil cooled transformers, cast resintransformers, isolating transformers, instrument transformers, currenttransformers, potential transformers, pulse transformers, air-coretransformers, ferrite-core transformers, transmission-line transformers,balun audio transformers, loudspeaker transformers, output transformers,small signal transformers, interstage coupling transformers, hedgehog orvariocoupler transformers.

Pulsing potentials in the power supply of the production units can alsobe built in. Lack of filter capacitors in the rectified power supply cancause the voltages to drop to zero a predetermined amount of times persecond. For example, at 60 Hz the voltage can spike 120 times persecond, resulting in a hard spike when the alternating current in thehouse power lines changes polarity. This hard spike, under Fouriertransform, can emit a large bandwidth of frequencies. In essence, thevoltage is varying from high potential to zero 120 times a second. Inother embodiments, the voltage can vary from high potential to zeroabout 1,000 times a second, about 500 times a second, about 200 times asecond, about 150 times a second, about 120 times a second, about 100times a second, about 80 times a second, about 50 times a second, about40 times a second, about 20 times a second, between about 200 times asecond and about 20 times a second, between about 150 times a second andabout 100 times a second, at least about 100 times a second, at leastabout 50 times a second, or at least about 120 times a second. Thispower modulation can allow the electrodes sample all voltages and alsoprovides enough frequency bandwidth to excite resonances in the formingmolecules themselves. The time at very low voltages can also provide anenvironment of low electric fields where ions of similar charge can comewithin close proximity to the electrodes. All of these factors togethercan provide a possibility for the formation of stable complexes capableof generating and preserving ROS free radicals. In one embodiment, thepulsing potentials can vary based on the desired functional parametersand capabilities of the apparatus and equipment and to that end can varyfrom very high potentials to low potentials and from very highfrequencies to very low frequencies. In one embodiment, the voltagepotential must go down to zero periodically. The voltage can go to 0 Vas many times per second as is physically possible. In some embodiments,the voltage is 0 V between 100 and 200 times per second. In a preferredembodiment, the voltage goes down to 0 V 120 times per second.

In some embodiments, there is no limit to the how high the voltagepotential can go. For example, the voltage potential can pulse from 0Vto 40V. In some embodiments, the voltage range can change or be changedso that the range changes as often or as little as desired within anygiven amount of time.

This pulsing waveform model can be used to stabilize superoxides,hydroxyl radicals and OOH* from many different components and is notlimited to any particular variable such as voltage, amps, frequency,flux (current density) or current. The variables are specific to thecomponents used. For example, water and NaCl can be combined whichprovide molecules and ions in solution. A 60 Hz current can be used,meaning that there are 60 cycles/120 spikes in the voltage (V) persecond or 120 times wherein the V is zero each second. When the V goesdown to zero it is believe that the 0 V allows for ions to driftapart/migrate and reorganize before the next increase in V. It istheorized that this spiking in V allows for and promotes a variablerange of frequencies influencing many different types of compoundsand/or ions so that this process occurs.

In one embodiment, periodic moments of 0 volts are required. Again, whenthe V goes down to zero it is believe that the 0 V allows for ions todrift apart/migrate and reorganize before the next increase in V.Therefore, without being bound to theory, it is believed that thismigration of ions facilitates the 1st, 2nd, and 3rd generations ofspecies as shown in FIG. 2. Stabilized superoxides, such as O₂*⁻, areproduced by this method.

In another embodiment, the V is always either zero or a positivepotential.

Diodes may also be used. The V may drop to zero as many times per secondas the frequency is adjusted. As the frequency is increased the numberof times the V drops is increased.

When the ions are affected by the electricity from the electrodes, theychange. Without being bound by theory, it is believed that theelectricity alters the state of some of the ions/compounds. Thisalteration results in the pushing of electrons out of their originalorbit and/or spin state into a higher energy state and/or a single spinstate. This electrolysis provides the energy to form free radicals whichare ultimately formed during a multi-generational cycling of reactantsand products during the electrolysis process. In other words, compoundsand/or ions are initially electrolyzed so that the products that areformed are then themselves reacted with other compounds and/or ionsand/or gas to form a second generation of reactants and products. Thisgenerational process then happens again so that the products from thesecond generation react with other compounds and/or ions in solutionwhen the voltage spikes again.

The redox potential can be about 840 mV.

The frequency can be from 1 Hz to infinity or to 100 MHz. Preferably,the frequency is from 20 Hz to 100 Hz. More preferably, the frequency isfrom 40 Hz to 80 Hz. Most preferably, the frequency is 60 Hz.

In another embodiment, the frequency changes during the course of theelectrolyzing process. For example, the frequency at any given moment isin the range from 20 Hz to 100 Hz. In another more preferred embodiment,the frequency at any given moment is in the range from 40 Hz to 80 Hz.

Again referencing FIG. 2, FIG. 2 illustrates an example diagram of thegeneration of various molecules at the electrodes, the molecules writtenbetween the electrodes depict the initial reactants and those on theoutside of the electrodes depict the molecules/ions produced at theelectrodes and their electrode potentials. The diagram is broken intogenerations where each generation relies on the products of thesubsequent generations.

The end products of this electrolytic process can react within thesaline solution to produce many different chemical entities. Thecompositions described herein can include one or more of these chemicalentities. These end products can include, but are not limited tosuperoxides (O₂*⁻, HO₂*), hypochlorites (OCl⁻, HOCl, NaOCl),hypochlorates (HClO₂, ClO₂, HClO₃, HClO₄), oxygen derivatives (O₂, O₃,O₄*⁻, 1O), hydrogen derivatives (H₂, H⁻), hydrogen peroxide (H₂O₂),hydroxyl free radical (OH*⁻), ionic compounds (Na⁺, Cl⁻, H⁺, OH⁻, NaCl,HCl, NaOH), chlorine (Cl₂), and water clusters (n*H₂O-induced dipolarlayers around ions), several other variations.

In one embodiment, the composition can include at least one species suchas O₂, H₂, Cl₂, Off, HOCl, NaOCl, HClO₂, ClO₂, HClO₃, HClO₄, H₂O₂, Na⁺,Cl⁻, H⁺, H, OH⁻, O₃, O₄*, 1O, OH*⁻, HOCl—O₂*⁻, HOCl—O₃, O₂*, HO₂*, NaCl,HCl, NaOH, water clusters, or a combination thereof.

In one embodiment, the composition can include at least one species suchas H2, Cl2, OCI—, HOCI, NaOCl, HClO₂, ClO₂, HClO₃, HClO₄, H₂O₂, O₃, O₄*,1O₂, OH*⁻, HOCl—O₂*⁻, HOCl—O₃, O₂*, HO₂*, water clusters, or acombination thereof.

In one embodiment, the composition can include at least one species suchas HClO₃, HClO₄, H₂O₂, O₃, O₄*, 1O₂, OH*—, HOCl—O₂*—, HOCl—O₃, O₂*,HO₂*, water clusters, or a combination thereof.

In one embodiment, the composition can include at least O₂*— and HOCl.

In one embodiment, the composition can include O₂. In one embodiment,the composition can include H₂. In one embodiment, the composition caninclude Cl₂. In one embodiment, the composition can include OCl⁻. In oneembodiment, the composition can include HOCl. In one embodiment, thecomposition can include NaOCl. In one embodiment, the composition caninclude HClO₂. In one embodiment, the composition can include CI02. Inone embodiment, the composition can include HClO₃. In one embodiment,the composition can include HClO₄. In one embodiment, the compositioncan include H₂O₂. In one embodiment, the composition can include Na⁺. Inone embodiment, the composition can include Cl⁻. In one embodiment, thecomposition can include H. In one embodiment, the composition caninclude H. In one embodiment, the composition can include OH—. In oneembodiment, the composition can include O₃. In one embodiment, thecomposition can include O₄*. In one embodiment, the composition caninclude 102. In one embodiment, the composition can include OH*—. In oneembodiment, the composition can include HOCl—O₂*—. In one embodiment,the composition can include HOCl—O₃. In one embodiment, the compositioncan include O₂*—. In one embodiment, the composition can include HO₂*.In one embodiment, the composition can include NaCl. In one embodiment,the composition can include HCl. In one embodiment, the composition caninclude NaOH. In one embodiment, the composition can include waterclusters. Embodiments can include combinations thereof.

In some embodiments, hydroxyl radicals can be stabilized in thecomposition by the formation of radical complexes. The radical complexescan be held together by hydrogen bonding. Another radical that can bepresent in the composition is an OOH* radical. Still other radicalcomplexes can include a nitroxyl-peroxide radical (HNO—HOO*) and/or ahypochlorite-peroxide radical (HOCl—HOO*).

The composition is stable which means, among other things, that theactive agents are present, measurable or detected throughout thelifespan of the composition. In one embodiment, the active agent(s) oractive ingredient(s) are superoxides and/or hydroxyl radicals. Forexample, in some embodiments the composition may comprise at least somepercentage of the active ingredient(s) is present in the compositionafter a certain number of years, such as wherein at least 95% of theactive ingredient(s) is present in the composition after 2 years,wherein at least 90% of the active ingredient(s) is present in thecomposition after 3 years, wherein at least 85% of the activeingredient(s) is present in the composition after 4 years, wherein atleast 80% of the active ingredient(s) is present in the compositionafter 5 years, wherein at least 75% of the active ingredient(s) ispresent in the composition after 6 years, wherein at least 70% of theactive ingredient(s) is present in the composition after 7 years,wherein at least 65% of the active ingredient(s) is present in thecomposition after 8 years, wherein at least 60% of the activeingredient(s) is present in the composition after 9 years, wherein atleast 55% of the active ingredient(s) is present in the compositionafter 10 years and the like.

Stable oxygen radicals can remain stable for about 3 months, about 6months, about 9 months, about 12 months, about 15 months, about 18months, about 21 months, between about 9 months and about 15 months,between about 12 months and about 18 months, at least about 9 months, atleast about 12 months, at least about 15 months, at least about 18months, about 24 months, about 30 months, about 50 months, about 100months, about 200 months, about 300 months, about 400 months, about 500months, about 1000 months, about 2000 months, or longer.

Stable oxygen radicals can be substantially stable. Substantially stablecan mean that the stable oxygen radical can remain at a concentrationgreater than about 75% relative to the concentration on day 1 (day 1meaning on the day or at the time it was produced), greater than about80%, greater than about 85%, greater than about 90%, greater than about95%, greater than about 96%, greater than about 97%, greater than about98%, or greater than about 99% over a given time period as describedabove. For example, in one embodiment, the stable oxygen is at aconcentration greater than about 95% relative to day 1 for at least 1year. In another embodiment, the at least one oxygen radical is at aconcentration greater than about 98% for at least 1 year.

Stable can mean that the stable oxygen radical can remain at aconcentration greater than about 75% relative to the concentration onday 1 or the day is was produced, greater than about 80% relative to theconcentration on day 1 or the day is was produced, greater than about85% relative to the concentration on day 1 or the day is was produced,greater than about 90% relative to the concentration on day 1 or the dayis was produced, greater than about 95% relative to the concentration onday 1 or the day is was produced, greater than about 96% relative to theconcentration on day 1 or the day is was produced, greater than about97% relative to the concentration on day 1 or the day is was produced,greater than about 98% relative to the concentration on day 1 or the dayis was produced, or greater than about 99% relative to the concentrationon day 1 or the day is was produced over a given time period asdescribed above. For example, in one embodiment, the stable oxygen is ata concentration greater than about 95% relative to day 1 for at least 1year. In another embodiment, the at least one oxygen radical is at aconcentration greater than about 98% for at least 1 year.

Stability as used herein can also refer to the amount of a particularspecies when compared to a reference sample. In some embodiments, thereference sample can be made in 1 L vessels with 0.9% isotonic solutionelectrolyzed with 3 A at 40° F., for 3 min. In another embodiment, thereference sample can be made according to a process as otherwisedescribed herein. The reference standard can also be bottled directlyoff the processing line as a “fresh” sample.

In other embodiments, the at least one oxygen radical is greater thanabout 86% stable for at least 4 years, greater than about 79% stable forat least 6 years, greater than about 72% stable for at least 8 years,greater than about 65% stable for at least 10 years, or 100% stable forat least 20 years.

In still other embodiments, the at least one oxygen radical is greaterthan about 95% stable for at least 2 years, at least 3 years, at least 4years, at least 5 years, at least 6 years, at least 7 years, at least 8years, at least 9 years, at least 10 years, at least 15 years, or atleast 20 years. In still other embodiments, the at least one oxygenradical is greater than about 96% stable for at least 2 years, at least3 years, at least 4 years, at least 5 years, at least 6 years, at least7 years, at least 8 years, at least 9 years, at least 10 years, at least15 years, or at least 20 years. In still other embodiments, the at leastone oxygen radical is greater than about 97% stable for at least 2years, at least 3 years, at least 4 years, at least 5 years, at least 6years, at least 7 years, at least 8 years, at least 9 years, at least 10years, at least 15 years, or at least 20 years. In still otherembodiments, the at least one oxygen radical is greater than about 98%stable for at least 2 years, at least 3 years, at least 4 years, atleast 5 years, at least 6 years, at least 7 years, at least 8 years, atleast 9 years, at least 10 years, at least 15 years, or at least 20years. In still other embodiments, the at least one oxygen radical isgreater than about 99% stable for at least 2 years, at least 3 years, atleast 4 years, at least 5 years, at least 6 years, at least 7 years, atleast 8 years, at least 9 years, at least 10 years, at least 15 years,or at least 20 years. In still other embodiments, the at least oneoxygen radical is 100% stable for at least 2 years, at least 3 years, atleast 4 years, at least 5 years, at least 6 years, at least 7 years, atleast 8 years, at least 9 years, at least 10 years, at least 15 years,or at least 20 years.

The stability of oxygen radicals can also be stated as a decay rate overtime. Decay of superoxides is described in Ong, Ta-Chung, “DetailedMechanistic and Optimization of the Photochemical Production Method ofSuperoxide” (2007) which is incorporated herein in its entirety.Substantially stable can mean a decay rate less than 1% per month, lessthan 2% per month, less than 3% per month, less than 4% per month, lessthan 5% per month, less than 6% per month, less than 10% per month, lessthan 3% per year, less than 4% per year, less than 5% per year, lessthan 6% per year, less than 7% per year, less than 8% per year, lessthan 9% per year, less than 10% per year, less than 15% per year, lessthan 20% per year, less than 25% per year, between less than 3% permonth and less than 7% per year.

In other embodiments, stability can be expressed as a half-life. Ahalf-life of the stable oxygen radical can be about 6 months, about 1year, about 2 years, about 3 years, about 4 years, about 5 years, about10 years, about 15 years, about 20 years, about 24 years, about 30years, about 40 years, about 50 years, greater than about 1 year,greater than about 2 years, greater than about 10 years, greater thanabout 20 years, greater than about 24 years, between about 1 year andabout 30 years, between about 6 years and about 24 years, or betweenabout 12 years and about 30 years.

Reactive species' concentrations in the life enhancing solutions,detected by fluorescence photo spectroscopy, may not significantlydecrease in time. Mathematical models show that bound HOCl—*O₂ ⁻complexes are possible at room temperature. Molecular complexes canpreserve volatile components of reactive species. For example, reactivespecies concentrations in whole blood as a result of molecular complexesmay prevent reactive species degradation over time.

Reactive species can be further divided into “reduced species” (RS) and“reactive oxygen species” (ROS). Reactive species can be formed fromwater molecules and sodium chloride ions when restructured through aprocess of forced electron donation. Electrons from lower molecularenergy configurations in the salinated water may be forced into higher,more reactive molecular configurations. The species from which theelectron was taken can be “electron hungry” and is called the RS and canreadily become an electron acceptor (or proton donor) under the rightconditions. The species that obtains the high-energy electron can be anelectron donor and is called the ROS and may energetically release theseelectrons under the right conditions.

When an energetic electron in ROS is unpaired it is called a “radical”.ROS and RS can recombine to neutralize each other by the use of acatalytic enzyme. Three elements, (1) enzymes, (2) electron acceptors,and (3) electron donors can all be present at the same time and locationfor neutralization to occur.

Depending on the parameters used to produce the composition, differentcomponents can be present at different concentrations. In someembodiments, the composition can include about 0.1 ppt (part pertrillion), about 0.5 ppt, about 1 ppt, about 1.5 ppt, about 2 ppt, about2.5 ppt, about 3 ppt, about 3.5 ppt, about 4 ppt, about 4.5 ppt, about 5ppt, about 6 ppt, about 7 ppt, about 8 ppt, about 9 ppt, about 10 ppt,about 20 ppt, about 50 ppt, about 100 ppt, about 200 ppt, about 400 ppt,about 1,000 ppt, between about 0.1 ppt and about 1,000 ppt, betweenabout 0.1 ppt and about 100 ppt, between about 0.1 ppt and about 10 ppt,between about 2 ppt and about 4 ppt, at least about 0.1 ppt, at leastabout 2 ppt, at least about 3 ppt, at most about 10 ppt, or at mostabout 100 ppt of OCI—. In some embodiments, OCl⁻ can be present at 3ppt. In other embodiments, OCl⁻ can be present at 1 to 100 ppm or from10 to 30 ppm or from 16 to 24 ppm. In particular embodiments, OCl⁻ ispresent at 16 ppm, 17 ppm, 18 ppm, 19 ppm, 20 ppm, 21 ppm, 22 ppm, 23ppm, 24 ppm or 25 ppm. In other embodiments, OCl⁻ can be the predominantchlorine containing species in the composition.

In order to determine the relative concentrations and rates ofproduction of each of these during electrolysis, certain generalchemical principles can be helpful:

1) A certain amount of Gibbs free energy is required for construction ofthe molecules; Gibbs free energy is proportional to the differences inelectrode potentials listed in FIG. 2. Reactions with large energyrequirements are less likely to happen, for example an electrodepotential of −2.71V (compared to Hydrogen reduction at 0.00 V) isrequired to make sodium metal:

Na++1e ⁻→Na_((s))

Such a large energy difference requirement makes this reaction lesslikely to happen compared to other reactions with smaller energyrequirements. Electron(s) from the electrodes may be preferentially usedin the reactions that require lesser amounts of energy, such as theproduction of hydrogen gas.

2) Electrons and reactants are required to be at the same micro-localityon the electrodes. Reactions that require several reactants may be lesslikely to happen, for example:

Cl₂+6H₂O→10e ⁻+2ClO₃ ⁻+12H⁺

requires that 6 water molecules and a Cl₂ molecule to be at theelectrode at the same point at the same time and a release of 10electrons to simultaneously occur. The probability of this happeninggenerally is smaller than other reactions requiring fewer and moreconcentrated reactants to coincide, but such a reaction may still occur.

3) Reactants generated in preceding generations can be transported ordiffuse to the electrode where reactions happen. For example, dissolvedoxygen (O₂) produced on the anode from the first generation can betransported to the cathode in order to produce superoxides and hydrogenperoxide in the second generation. Ions can be more readily transported:they can be pulled along by the electric field due to their electriccharge. In order for chlorates, to be generated, for example, HClO₂ canfirst be produced to start the cascade, restrictions for HClO₂production can also restrict any subsequent chlorate production. Lowertemperatures can prevent HClO₂ production.

Stability and concentration of the above products can depend, in somecases substantially, on the surrounding environment. The formation ofcomplexes and water clusters can affect the lifetime of the moieties,especially the free radicals.

In a pH-neutral aqueous solution (pH around 7.0) at room temperature,superoxide free radicals (O₂*⁻) have a half-life of tens of millisecondsand dissolved ozone (O₃) has a half-life of about 20 min. Hydrogenperoxide (H₂O₂) is relatively long-lived in neutral aqueousenvironments, but this can depend on redox potentials and UV light.Other entities such as HCl and NaOH rely on acidic or basicenvironments, respectively, in order to survive. In pH-neutralsolutions, H⁺ and OH⁻ ions have concentrations of approximately 1 partin 10,000,000 in the bulk aqueous solution away from the electrodes. H⁻and 1O can react quickly. The stability of most of these moietiesmentioned above can depend on their microenvironment.

Superoxides and ozone can form stable Van de Waals molecular complexeswith hypochlorites. Clustering of polarized water clusters aroundcharged ions can also have the effect of preservinghypochlorite-superoxide and hypochlorite-ozone complexes. Such complexescan be built through electrolysis on the molecular level on catalyticsubstrates, and may not occur spontaneously by mixing togethercomponents. Hypochlorites can also be produced spontaneously by thereaction of dissolved chlorine gas (Cl₂) and water. As such, in aneutral saline solution the formation of one or more of the stablemolecules and complexes may exist: dissolved gases: O₂, H₂, Cl₂;hypochlorites: Off, HOCl, NaOCl; hypochlorates: HClO₂, ClO₂, HClO₃,HClO₄; hydrogen peroxide: H₂O₂; ions: Na⁺, Cl⁻, H⁺, H⁻, OH⁻; ozone: O₃,O₄*—; singlet oxygen: 1O; hydroxyl free radical: OH*⁻; superoxidecomplexes: HOCl—O₂*—; and ozone complexes: HOCl—O₃. One or more of theabove molecules can be found within the compositions and compositiondescribed herein.

A complete quantum chemical theory can be helpful because production iscomplicated by the fact that different temperatures, electrodegeometries, flows and ion transport mechanisms and electrical currentmodulations can materially change the relative/absolute concentrationsof these components, which could result in producing different distinctcompositions. As such, the selection of production parameters can becritical. The amount of time it would take to check all the variationsexperimentally may be prohibitive.

The chlorine concentration of the electrolyzed solution can be about 5ppm, about 10 ppm, about 15 ppm, about 20 ppm, about 21 ppm, about 22ppm, about 23 ppm, about 24 ppm, about 25 ppm, about 26 ppm, about 27ppm, about 28 ppm, about 29 ppm, about 30 ppm, about 31 ppm, about 32ppm, about 33 ppm, about 34 ppm, about 35 ppm, about 36 ppm, about 37ppm, about 38 ppm, less than about 38 ppm, less than about 35 ppm, lessthan about 32 ppm, less than about 28 ppm, less than about 24 ppm, lessthan about 20 ppm, less than about 16 ppm, less than about 12 ppm, lessthan about 5 ppm, between about 30 ppm and about 34 ppm, between about28 ppm and about 36 ppm, between about 26 ppm and about 38 ppm, betweenabout 20 ppm and about 38 ppm, between about 5 ppm and about 34 ppm,between about 10 ppm and about 34 ppm, or between about 15 ppm and about34 ppm. In one embodiment, the chlorine concentration is about 32 ppm.In another embodiment, the chlorine concentration is less than about 41ppm.

In some embodiments, the chloride species can be present from 1400 to1650 ppm. In a particular embodiment, the chloride species can bepresent from 1400 to 1500 ppm or from 1500 to 1600 ppm or from 1600 to1650 ppm. In other embodiments, the chloride anion can be present in anamount that is predetermined based on the amount of NaCl added to theinitial solution.

In some embodiments, the sodium species can be present from 1000 to 1400ppm. In a particular embodiment, the sodium species can be present from1100 to 1200 ppm or from 1200 to 1300 ppm or from 1300 to 1400 ppm. Forexample, the sodium species can be present at 1200 ppm. In otherembodiments, the sodium anion can be present in an amount that ispredetermined based on the amount of NaCl added to the initial solution.

The saline concentration in the electrolyzed solution can be about 0.10%w/v, about 0.11% w/v, about 0.12% w/v, about 0.13% w/v, about 0.14% w/v,about 0.15% w/v, about 0.16% w/v, about 0.17% w/v, about 0.18% w/v,about 0.19% w/v, about 0.20% w/v, about 0.30% w/v, about 0.40% w/v,about 0.50% w/v, about 0.60% w/v, about 0.70% w/v, between about 0.10%w/v and about 0.20% w/v, between about 0.11% w/v and about 0.19% w/v,between about 0.12% w/v and about 0.18% w/v, between about 0.13% w/v andabout 0.17% w/v, or between about 0.14% w/v and about 0.16% w/v.

The composition generally can include electrolytic and/or catalyticproducts of pure saline that mimic redox signaling molecularcompositions of the native salt water compounds found in and aroundcells. The composition can be fine-tuned to mimic or mirror molecularcompositions of different biological media. The composition can havereactive species other than chlorine present. As described, speciespresent in the compositions and compositions described herein caninclude, but are not limited to O₂, H₂, Cl₂, OCl⁻, HOCl, NaOCl, HClO₂,ClO₂, HClO₃, HClO₄, H₂O₂, Na⁺, Cl, H⁺, H⁻, OH⁻, O₃, O₄*, 1O, OH*⁻,HOCl—O₂*—, HOCl—O₃, O₂*, HO₂*, NaCl, HCl, NaOH, and water clusters:n*H₂O-induced dipolar layers around ions, several variations.

In some embodiments, substantially no organic material is present in thecompositions described. Substantially no organic material can be lessthan about 0.1 ppt, less than about 0.01 ppt, less than about 0.001 pptor less than about 0.0001 ppt of total organic material.

The composition can be stored and bottled as needed to ship toconsumers. The composition can have a shelf life of about 5 days, about30 days, about 3 months, about 6 months, about 9 months, about 1 year,about 1.5 years, about 2 years, about 3 years, about 5 years, about 10years, at least about 5 days, at least about 30 days, at least about 3months, at least about 6 months, at least about 9 months, at least about1 year, at least about 1.5 years, at least about 2 years, at least about3 years, at least about 5 years, at least about 10 years, between about5 days and about 1 year, between about 5 days and about 2 years, betweenabout 1 year and about 5 years, between about 90 days and about 3 years,between about 90 days and about 5 year, or between about 1 year andabout 3 years.

Quality Assurance testing can be done on every batch before the batchcan be approved for bottling or can be performed during or afterbottling. A 16 oz. sample bottle can be taken from each complete batchand analyzed. Determinations for presence of contaminants such as heavymetals or chlorates can be performed. Then pH, Free and Total Chlorineconcentrations and reactive molecule concentrations of the activeingredients can be analyzed by fluorospectroscopy methods. These resultscan be compared to those of a standard solution which is also testedalongside every sample. If the results for the batch fall within acertain range relative to the standard solution, it can be approved. Achemical chromospectroscopic MS analysis can also be run on randomsamples to determine if contaminants from the production process arepresent.

In some embodiments the gel or hydrogel can be administered and/orapplied topically to a user. The topical product can be administeredand/or applied to the user in ounce units such as from 0.5 oz. to 20 oz.or as desired by the user. When applied to the user, it can be appliedonce, twice, three times, four times or more a day. Each application tothe user can be about 1 oz., about 2 oz., about 3 oz., about 4 oz.,about 5 oz., about 6 oz., about 7 oz., about 8 oz., about 9 oz., about10 oz., about 11 oz., about 12 oz., about 16 oz., or about 20 oz. In oneembodiment, the composition can be applied to the user at a rate ofabout 4 oz. twice a day.

In other embodiments, the administration and/or application to the usercan be acute or long term. For example, the composition can beadministered to the user for a day, a week, a month, a year or longer.In other embodiments, the composition can simply be applied to the useras needed.

Example 1

FIG. 3 illustrates a plan view of a process and system for producing aredox signaling composition comprising redox signaling agents accordingto the present description. One skilled in the art understands thatchanges can be made to the system to alter the composition, and thesechanges are within the scope of the present description.

Incoming water 202 can be subjected to reverse osmosis system 204 at atemperature of about 15-20° C. to achieve purified water 206 with about8 ppm of total dissolved solids. Purified water 206, is then fed at atemperature of about 15-20° C. into distiller 208 and processed toachieve distilled water 210 with about 0.5 ppm of total dissolvedsolids. Distilled water 210 can then be stored in tank 212.

FIG. 4 illustrates an example system for preparing water for furtherprocessing into a therapeutic composition. System 300 can include awater source 302 which can feed directly into a carbon filter 304. Afteroils, alcohols, and other volatile chemical residuals and particulatesare removed by carbon filter 304, the water can be directed to resinbeds within a water softener 306 which can remove dissolved minerals.Then, as described above, the water can pass through reverse osmosissystem 204 and distiller 208.

Referring again to FIG. 3, distilled water 210 can be gravity fed asneeded from tank 212 into saline storage tank cluster 214 using line216. Saline storage tank cluster 214 in one embodiment can includetwelve tanks 218. Each tank 218 can be filled to about 1,300 gallonswith distilled water 210. A handheld meter can be used to test distilledwater 210 for salinity.

Saline storage tank cluster 214 is then salted using a brine system 220.Brine system 220 can include two brine tanks 222. Each tank can have acapacity of about 500 gallons. Brine tanks 222 are filled to 475 gallonswith distilled water 210 using line 224 and then NaCl is added to thebrine tanks 222 at a ratio of about 537.5 g/gal of liquid. At thispoint, the water is circulated 226 in the brine tanks 222 at a rate ofabout 2,000 gal/h for about 4 days.

Prior to addition of brine to tanks 218, the salinity of the water intanks 218 can be tested using a handheld conductivity meter such as anYSI ECOSENSE® ecp300 (YSI Inc., Yellow Springs, Ohio). Any correctionsbased on the salinity measurements can be made at this point. Brinesolution 228 is then added to tanks 218 to achieve a salt concentrationof about 10.75 g/gal. The salted water is circulated 230 in tanks 218 ata rate of about 2,000 gal/hr for no less than about 72 hours. Thiscirculation is performed at room temperature. A handheld probe can againbe used to test salinity of the salinated solution. In one embodiment,the salinity is about 2.8 ppt.

In one method for filling and mixing the salt water in the brine holdingtanks, the amount of liquid remaining in the tanks is measured. Theamount of liquid remaining in a tank is measured by recording the heightthat the liquid level is from the floor that sustains the tank, incentimeters, and referencing the number of gallons this heightrepresents. This can be done from the outside of the tank if the tank issemi-transparent. The initial liquid height in both tied tanks can alsobe measured. Then, after ensuring that the output valve is closed,distilled water can be pumped in. The amount of distilled water that isbeing pumped into a holding tank can then be calculated by measuring therise in liquid level: subtracting the initial height from the filledheight and then multiplying this difference by a known factor.

The amount of salt to be added to the tank is then calculated bymultiplying 11 grams of salt for every gallon of distilled water thathas been added to the tank. The salt can be carefully weighed out anddumped into the tank.

The tank is then agitated by turning on the recirculation pump and thenopening the top and bottom valves on the tank. Liquid is pumped from thebottom of the tank to the top. The tank can be agitated for three daysbefore it may be ready to be processed.

After agitating the tank for more than 6 hours, the salinity is checkedwith a salinity meter by taking a sample from the tank and testing it.Salt or water can be added to adjust the salinity within the tanks. Ifeither more water or more salt is added then the tanks are agitated for6 more hours and tested again. After about three days of agitation, thetank is ready to be processed.

Salinated water 232 is then transferred to cold saline tanks 234. In oneembodiment, four 250 gal tanks are used. The amount of salinated water232 moved is about 1,000 gal. A chiller 236 such as a 16 ton chiller isused to cool heat exchangers 238 to about 0-50 C. The salinated water iscirculated 240 through the heat exchangers which are circulated withpropylene glycol until the temperature of the salinated water is about4.5-5.8° C. Chilling the 1,000 gal of salinated water generally takesabout 6-8 hr.

Cold salinated water 242 is then transferred to processing tanks 244. Inone embodiment, eight tanks are used and each can have a capacity ofabout 180 gal. Each processing tank 244 is filled to about 125 gal for atotal of 1,000 gal. Heat exchangers 246 are again used to chill the coldsalinated water 242 added to processing tanks 244. Each processing tankcan include a cylinder of chilling tubes and propylene glycol can becirculated. The heat exchangers can be powered by a 4-5 ton chiller 248.The temperature of cold salinated water 242 can remain at 4.5-5.8° C.during processing.

Prior to transferring aged salt water to processing tanks, the aged saltwater can be agitated for about 30 minutes to sufficiently mix the agedsalt water. Then, the recirculation valves can then be closed, theappropriate inlet valve on the production tank is opened, and the tankfilled so that the salt water covers the cooling coils and comes up tothe fill mark (approximately 125 gallons).

Once the aged salt water has reached production temperature, the pump isturned off but the chiller left on. The tank should be adequatelyagitated or re-circulated during the whole duration of electrochemicalprocessing and the temperature should remain constant throughout.

Each processing tank 244 includes electrode 250. Electrodes 250 can be 3inches tall circular structures formed of titanium and plated withplatinum. Electrochemical processing of the cold salinated water can berun for 8 hr. A power supply 252 is used to power the eight electrodes(one in each processing tank 244) to 7 amps each for a total of 56 amps.The cold salinated water is circulated 254 during electrochemicalprocessing at a rate of about 1,000 gal/h.

An independent current meter can be used to set the current to around7.0 Amps. Attention can be paid to ensure that the voltage does notexceed 12V and does not go lower than 9V. Normal operation can be about10 V. Alternatively, normal operation can be at 1V, 2V, 3V, 4V, 5V, 6V,7V, 8V, 9V, 10V, 11V or 12V.

A run timer can be set for a prescribed time (about 4.5 to 5 hours).Each production tank can have its own timer and/or power supply.Electrodes should be turned off after the timer has expired.

The production tanks can be checked periodically. The temperature and/orelectrical current can be kept substantially constant. At the beginning,the electrodes can be visible from the top, emitting visible bubbles.After about 3 hours, small bubbles of un-dissolved oxygen can startbuilding up in the tank as oxygen saturation occurs, obscuring the viewof the electrodes. A slight chlorine smell can be normal.

After the 8 hour electrochemical processing is complete, life enhancingwater 256 has been created with a pH of about 6.8-8.2 and 32 ppm ofchlorine. The composition 256 is transferred to storage tanks 258. Insome embodiments, the product ASEA is made by the process of thisExample 1.

Example 2

Characterization of a Solution Produced as Described in Example 1

A composition produced as described in Example 1 was analyzed using avariety of different characterization techniques. ICP/MS and 35CI NMRwere used to analyze and quantify chlorine content. Headspace massspectrometry analysis was used to analyze adsorbed gas content in thecomposition. 1H NMR was used to verify the organic matter content in thecomposition. 31P NMR and EPR experiments utilizing spin trap moleculeswere used to explore the composition for free radicals.

The composition was received and stored at about 4° C. when not beingused.

Chlorine NMR

Sodium hypochlorite solutions were prepared at different pH values. 5%sodium hypochlorite solution had a pH of 12.48. Concentrated nitric acidwas added to 5% sodium hypochlorite solution to create solutions thatwere at pH of 9.99, 6.99, 5.32, and 3.28. These solutions were thenanalyzed by NMR spectroscopy. The composition had a measured pH of 8.01and was analyzed directly by NMR with no dilutions.

NMR spectroscopy experiments were performed using a 400 MHz Brukerspectrometer equipped with a BBO probe. 35Cl NMR experiments wereperformed at a frequency of 39.2 MHz using single pulse experiments. Arecycle delay of 10 seconds was used, and 128 scans were acquired persample. A solution of NaCl in water was used as an external chemicalshift reference. All experiments were performed at room temperature.

35Cl NMR spectra were collected for NaCl solution, NaClO solutionsadjusted to different pH values, and the composition. FIG. 5 illustratesa Cl³⁵ spectrum of NaCl, NaClO solution at a pH of 12.48, and thecomposition. The chemical shift scale was referenced by setting the Cl⁻peak to 0 ppm. NaClO solutions above a pH=7 had identical spectra with apeak at approximately 5.1 ppm. Below pH of 7.0, the CIO— peakdisappeared and was replaced by much broader, less easily identifiablepeaks. The composition was presented with one peak at approximately 4.7ppm, from CIO— in the composition. This peak was integrated to estimatethe concentration of CIO— in the composition, which was determined to be2.99 ppt or 0.17 M of ClO⁻ in the composition.

Proton NMR

An ASEA sample was prepared by adding 550 μL of ASEA and 50 μL of 020(Cambridge Isotope Laboratories) to an NMR tube and vortexing the samplefor 10 seconds. 1H NMR experiments were performed on a 700 MHz Brukerspectrometer equipped with a QNP cryogenically cooled probe. Experimentsused a single pulse with pre-saturation on the water resonanceexperiment. A total of 1024 scans were taken. All experiments wereperformed at room temperature.

A 1H NMR spectrum of the composition was determined and is presented inFIG. 6. Only peaks associated with water were able to be distinguishedfrom this spectrum. This spectrum show that very little if any organicmaterial can be detected in the composition using this method.

Phosphorous NMR and Mass Spectrometry

DIPPMPO (5-(Diisopropoxyphosphoryl)-5-1-pyrroline-N-oxide) (VWR) sampleswere prepared by measuring about 5 mg of DIPPMPO into a 2 ml centrifugetube. This tube then had 550 μL of either the composition or water addedto it, followed by 50 μL of 020. A solution was also prepared with thecomposition but without DIPPMPO. These solutions were vortexed andtransferred to NMR tubes for analysis. Samples for mass spectrometryanalysis were prepared by dissolving about 5 mg of DIPPMPO in 600 μL ofthe composition and vortexing, then diluting the sample by adding 100 μLof sample and 900 μL of water to a vial and vortexing.

NMR experiments were performed using a 700 MHz Bruker spectrometerequipped with a QNP cryogenically cooled probe. Experiments performedwere a single 30° pulse at a 31P frequency of 283.4 MHz. A recycle delayof 2.5 seconds and 16384 scans were used. Phosphoric acid was used as anexternal standard. All experiments were performed at room temperature.

Mass spectrometry experiments were performed by directly injecting theASENDIPPMPO sample into a Waters/Synapt Time of Flight massspectrometer. The sample was directly injected into the massspectrometer, bypassing the LC, and monitored in both positive andnegative ion mode.

31P NMR spectra were collected for DIPPMPO in water, the compositionalone, and the composition with DIPPMPO added to it. An externalreference of phosphoric acid was used as a chemical shift reference.FIG. 7 illustrates a 31P NMR spectrum of DIPPMPO combined with thecomposition. The peak at 21.8 ppm was determined to be DIPPMPO and isseen in both the spectrum of DIPPMPO with the composition (FIG. 7) andwithout the composition (not pictured). The peak at 24.9 ppm is mostprobably DIPPMPO/OH. as determined in other DIPPMPO studies. This peakmay be seen in DIPPMPO mixtures both with and without the composition,but is detected at a much greater concentration in the solution with thecomposition. In the DIPPMPO mixture with the composition, there isanother peak at 17.9 ppm. This peak may be from another radical speciesin the composition such as OOH. or possibly a different radical complex.The approximate concentrations of spin trap complexes in the composition/DIPPMPO solution are as follows:

Solution Concentration DIPPMPO 36.6 mM DIPPMPO/OH• 241 μMDIPPMPO/radical 94 μM

Mass spectral data was collected in an attempt to determine thecomposition of the unidentified radical species. The mass spectrum showsa parent peak and fragmentation pattern for DIPPMPO with m/z peaks at264, 222, and 180, as seen in FIG. 8. FIG. 8 also shows peaks for theDIPPMPO/Na adduct and subsequent fragments at 286, 244, and 202 m/z.Finally, FIG. 8 demonstrates peaks for one DIPPMPO/radical complex withm/z of 329. The negative ion mode mass spectrum also had a correspondingpeak at m/z of 327. There are additional peaks at 349, 367, and 302 at alower intensity as presented in FIG. 8. None of these peaks could bepositively confirmed. However, there are possible structures that wouldresult in these mass patterns. One possibility for the peak generated at329 could be a structure formed from a radical combining with DIPPMPO.Possibilities of this radical species include a nitroxyl-peroxideradical (HNO—HOO.) that may have formed in the composition as a resultof reaction with nitrogen from the air. Another peak at 349 could alsobe a result of a DIPPMPO/radical combination. Here, a possibility forthe radical may be hypochlorite-peroxide (HOCl—HOO.). However, the smallintensity of this peak and small intensity of the corresponding peak of347 in the negative ion mode mass spectrum indicate this could be a verylow concentration impurity and not a compound present in the ASEAcomposition.

ICP/MS Analysis

Samples were analyzed on an Agilent 7500 series inductively-coupledplasma mass spectrometer (ICP-MS) in order to confirm the hypochloriteconcentration that was determined by NMR. A stock solution of 5% sodiumhypochlorite was used to prepare a series of dilutions consisting of 300ppb, 150 ppb, 75 ppb, 37.5 ppb, 18.75 ppb, 9.375 ppb, 4.6875 ppb,2.34375 ppb, and 1.171875 ppb in deionized Milli-Q water. Thesestandards were used to establish a standard curve.

Based on NMR hypochlorite concentration data, a series of dilutions wasprepared consisting of 164.9835 ppb, 82.49175 ppb, 41.245875 ppb,20.622937 ppb, 10.311468 ppb, and 5.155734 ppb. These theoretical valueswere then compared with the values determined by ICP-MS analysis. Theinstrument parameters were as follows:

Elements analyzed 3sc1 37CI′ # of points per mass 20 # of repetitions  5Total acquisition time 68.8 s Uptake speed 0.50 rps Uptake time 33 sStabilization time 40 s Tune No Gas Nebulizer flow rate 1 ml/min Torchpower 1500 W

The results of the ICP-MS analysis are as follows:

Dilution Measured Concentration (ppb) Concentration by NMR (ppb) 1 81 822 28 41 3 24 21 4 13 10 5 8 5

Dilutions were compared graphically to the ICP-MS signals and fit to alinear equation (R2=0.9522). Assuming linear behavior of the ICP-MSsignal, the concentration of hypochlorite in the composition wasmeasured to be 3.02 ppt. Concentration values were determined bycalculating the concentration of dilutions of the initial compositionand estimating the initial composition hypochlorite concentration to be3 ppt (as determined from 35CI NMR analysis). The ICP-MS data correlatewell with the 35CI NMR data, confirming a hypochlorite concentration ofroughly ⅓% (3 ppt). It should be noted that ICP-MS analysis is capableof measuring total chlorine atom concentration in solution, but notspecific chlorine species. The NMR data indicate that chlorinepredominantly exists as CIO− in the composition.

Gas Phase Quadrupole MS

Sample Prep

Three sample groups were prepared in triplicate for the analysis: 1)Milli-Q deionized water 2) the composition, and 3) 5% sodiumhypochlorite standard solution. The vials used were 20 ml headspacevials with magnetic crimp caps (GERSTEL). A small stir bar was placed ineach vial (VWR) along with 10 ml of sample. The vials were capped, andthen placed in a Branson model 5510 sonicator for one hour at 60° C.

The sonicator was set to degas which allowed for any dissolved gasses tobe released from the sample into the headspace. After degassing, thesamples were placed on a CTC PAL autosampler equipped with a heatedagitator and headspace syringe. The agitator was set to 750 rpm and 95°C. and the syringe was set to 75° C. Each vial was placed in theagitator for 20 min prior to injection into the instrument. A headspacevolume of 2.5 ml was collected from the vial and injected into theinstrument.

Instrument Parameters

The instrument used was an Agilent 7890A GC system coupled to an Agilent5975C El/Cl single quadrupole mass selective detector (MSD) set up forelectron ionization. The GC oven was set to 40° C. with the front inletand the transfer lines being set to 150° C. and 155° C. respectively.The carrier gas used was helium and it was set to a pressure of 15 PSI.

The MSD was set to single ion mode (SIM) in order to detect thefollowing analytes:

Analyte Mass Water 18 Nitrogen 28 Oxygen 32 Argon 40 Carbon dioxide 44Chlorine 70 Ozone 48

The ionization source temperature was set to 230° C. and the quadrupoletemperature was set to 150° C. The electron energy was set to 15 V.

Mass spectrometry data was obtained from analysis of the gas phaseheadspace of the water, the composition, and hypochlorite solution. Theraw area counts obtained from the mass spectrometer were normalized tothe area counts of nitrogen in order to eliminate any systematicinstrument variation. Both nitrogen and water were used as standardsbecause they were present in equal volumes in the vial with nitrogenoccupying the headspace and water being the solvent. It was assumed thatthe overall volume of water and nitrogen would be the same for eachsample after degassing. In order for this assumption to be correct, theratio of nitrogen to water should be the same for each sample. A cutoffvalue for the percent relative standard deviation (% RSD) of 5% wasused. Across all nine samples, a % RSD of 4.2 was observed. Of note,sample NaClO-3 appears to be an outlier, thus, when removed, the % RSDdrops to 3.4%.

FIGS. 9-11 illustrate oxygen/nitrogen, chlorine/nitrogen, andozone/nitrogen ratios. It appears that there were less of these gasesreleased from the composition than from either water or nitrogen. Itshould be noted that the signals for both ozone and chlorine were veryweak. Thus, there is a possibility that these signals may be due toinstrument noise and not from the target analytes.

FIG. 12 illustrates the carbon dioxide to nitrogen ratio. It appearsthat there may have been more carbon dioxide released from thecomposition than oxygen. However, it is possible that this may be due tobackground contamination from the atmosphere.

Based on the above, more oxygen was released from both water and sodiumhypochlorite than the composition.

EPR

Two different composition samples were prepared for EPR analysis. Thecomposition with nothing added was one sample. The other sample wasprepared by adding 31 mg of DIPPMPO to 20 ml of the composition (5.9mM), vortexing, and placing the sample in a 4° C. refrigeratorovernight. Both samples were placed in a small capillary tube which wasthen inserted into a normal 5 mm EPR tube for analysis.

EPR experiments were performed on a Bruker EMX 10/12 EPR spectrometer.EPR experiments were performed at 9.8 GHz with a centerfield position of3500 Gauss and a sweepwidth of 100 Gauss. A 20 mW energy pulse was usedwith modulation frequency of 100 kHz and modulation amplitude of 1G.Experiments used 100 scans. All experiments were performed at roomtemperature.

EPR analysis was performed on the composition with and without DIPPMPOmixed into the solution. FIG. 13 shows the EPR spectrum generated fromDIPPMPO mixed with the composition. The composition alone showed no EPRsignal after 100 scans (not presented). FIG. 13 illustrates an EPRsplitting pattern for a free electron. This electron appears to be splitby three different nuclei. The data indicate that this is acharacteristic splitting pattern of OH. radical interacting with DMPO(similar to DIPPMPO). This pattern can be described by 14N splitting thepeak into three equal peaks and 1H three bonds away splitting thatpattern into two equal triplets. If these splittings are the same, itleads to a quartet splitting where the two middle peaks are twice aslarge as the outer peaks. This pattern may be seen in FIG. 13 twice,with the larger peaks at 3457 and 3471 for one quartet and 3504 and 3518for the other quartet. In this case, the 14N splitting and the 1Hsplitting are both roughly 14G, similar to an OH* radical attaching toDMPO. The two quartet patterns in FIG. 13 are created by an additionalsplitting of 47G. This splitting is most likely from coupling to 31P,and similar patterns have been seen previously. The EPR spectrum in FIG.13 indicates that there is a DIPPMPO/OH. radical species in thesolution.

Example 3

This example describes a process and system for producing a redoxsignaling composition comprising redox signaling agents according to thepresent description. Electrolyzed fluid can be made in different typesof vessels as long as the proper power sourced is used. One example ofan apparatus that was used to make electrolyzed solution for treatingfungal infections is that referred to in FIGS. 14-18.

Referring first to FIG. 14, which is a perspective view of a firstpresently preferred embodiment generally represented at 100, includes apower supply 102 and a fluid receptacle represented at 104. The fluidreceptacle 104 includes a base 114 upon which is attached a fluid vessel116. The base 114 can preferably be fabricated from an insulativeplastic material. The fluid vessel 116 is preferably fabricated from aninert clear plastic material which is compatible with biologicalprocesses as available in the art.

A lid 118 is provided to cover the fluid vessel 116 and keepcontaminants out of the fluid vessel 116. A screen 120 is positioned toprevent foreign objects, which might accidentally fall into the fluidvessel 116, from falling to the bottom of the fluid vessel 116. Thesaline solution which is to be treated is placed into the fluid vessel116, and the lid 118 placed, for the necessary period of time afterwhich the electrolyzed saline solution can be withdrawn from the fluidvessel 116, for example into a syringe, for use. The fluid vessel 116 issealed at its bottom by a floor 124 which is attached to the interior ofthe base 114.

An electrode assembly, generally represented at 122, is attached to thefloor 124 so that any fluid in the fluid vessel is exposed to theelectrode assembly 122. The electrode assembly 122 is electricallyconnected to the power supply 102 via terminals 110 and 112 and cables106 and 108, respectively. The power supply 102 should deliver acontrolled voltage and current to the electrode assembly 122 when fluidis placed into the fluid vessel 116. The voltage and current applied tothe electrode assembly 122 will vary according to the fluid beingelectrolyzed. A control for setting and measuring the voltage 102A and acontrol for setting and measuring the current 1028 is provided in thepower supply. In accordance some embodiments, a low voltage of less thanabout 30 volts DC is used. Exemplary voltage and current values, and theadvantages which accrue when using the preferred voltage and currentvalues, will be explained shortly.

FIG. 15 is a top view of the electrode assembly 122 represented in FIG.14. The electrode assembly 122 preferably comprises a cylindrical innerelectrode 128 and a cylindrical outer electrode 126. The inner electrode128 is preferably solid or any hollow in the inner electrode is sealedso that fluid does not enter any such hollow. The cylindrical shape ofthe inner electrode 128 and the outer electrode 126 is preferred andresults in better performance than obtained with electrodes of othershapes, e.g., elongated flat panels.

The diameter A of the inner electrode 128 is preferably about one-halfinch but the diameter A of the inner electrode can be selected by thoseskilled in the art in accordance with the particular application for theelectrode using the information contained herein. The outer electrode126 should be of a generally cylindrical shape and preferably befabricated from titanium or niobium having a thickness (indicated at Bin FIG. 15) which ensures that the inner electrode is shielded frompotentially physical damage. As will be appreciated, titanium andniobium provide the advantage of resistance against corrosion whichfurther prevents the introduction of harmful substances into the fluidbeing electrolyzed.

Still referring to FIG. 15, the space, indicated at C, between the innerelectrode 128 and the outer electrode 126 does not exceed a maximumvalue. In contrast to previously available devices which separate theelectrodes by greater distances and then utilize higher voltages toobtain the desired electrolyzation, some embodiments keeps the electrodespacing small and obtains improved performance over other schemes. It ispreferred that the space between the inner electrode 128 and the outerelectrode 126 be not more than about one-half (½) inch; it is morepreferred that the space between the inner electrode 128 and the outerelectrode 126 be not more than about three-eighths (⅜) inch; and, it ismost preferred that the space between the inner electrode 128 and theouter electrode 126 be not more than about one-quarter (¼) inch.

Reference will next be made to FIG. 15A which is a side cross sectionalview of the electrode assembly taken along line 3-3 in FIG. 15. As seenin FIG. 15A, the outer electrode 126 extends above the inner electrode128 to provide improved electrical performance and physical protection.The outer electrode 126 is attached to the floor 124 by way of bolts130, which extend through bores provided in the floor 124, andaccompanying nuts. An electrical connection is made to the outerelectrode 126 by a lead 136 attached to the bolt and nut. The lead 136is attached to one of the terminals 110 or 112. Similarly, an electricalconnection is made to the inner electrode 128 by a lead 134 which isheld in place by a nut attached to a threaded stud extending from thebottom of the inner electrode and through a bore provided in the floor124. The lead 134 is attached to the remaining one of the terminals 110or 112. The leads 134 and 136 are kept insulated from any fluid which ispresent in the fluid vessel 116.

It is preferred that the inner electrode 128 function as the anode whilethe outer electrode function as the cathode when electrolyzing fluidsand the power supply 102 and the terminals 110 and 112 should beproperly arranged to carry this out.

It is recognized in the art that the anode is subject to destructiveforces during electrolysis. In the prior art, the anode of an electrodeassembly may dissolve to the point of being inoperative and may need tobe replaced very often. Critically, as the anode of an electrodeassembly dissolves, the metallic components of the anode are dispersedinto the fluid. If the fluid is a saline solution which will be used totreat physiological fluids, toxic substances dispersed into thesolution, such as the materials comprising the anode, may be harmful ordangerous to the person who expects to be benefited from the treatment.

Of all the possible materials for fabrication of the anode, the artrecognizes that platinum is the least likely to be dissolved when usedas an anode. Unfortunately, the cost of platinum precludes the use of ananode which consists entirely of platinum. Thus, it is common in the artto utilize another metal as a base for the anode with a layer ofplatinum being placed on surfaces which contact the fluid to beelectrolyzed.

Some embodiments advantageously utilize an inner electrode 128, i.e., ananode, which includes a base of titanium, and even more preferablyniobium (also known as columbium), upon which a layer of platinum isprovided wherever fluid contacts the anode. Significantly, niobium is arelatively good electrical conductor having a conductivity which isabout three times greater than the conductivity of titanium. Moreover,if the base metal is exposed to the fluid, such as if a pinhole defectdevelops, toxic products are not produced by the contact between niobiumand the fluid. Moreover, the high breakdown voltage in saline solutionof the oxide which forms when a niobium base receives a layer ofplatinum provides further advantages.

Upon a base of niobium, a layer of platinum is formed on the anode. Thelayer of platinum is preferably formed using a technique referred to inthe art as brush electrodeposition which can be carried out by thoseskilled in the art using the information set forth herein. Othertechniques can also be used to form the platinum layer, such as tank(immersion) electrodeposition, vapor deposition, and roll bonding, butbrush electrodeposition is preferred because of its superior adhesionand resulting less porosity than other economically comparabletechniques.

The thickness of the platinum layer is preferably greater than about0.02 mils and is most preferably greater than about 0.06 mils, and up toabout 0.20 mils. The combination of using niobium as a base for theanode of the electrode assembly and utilizing brush electrodepositionprovides that the platinum layer can be much thinner than otherwisepossible and still provide economical and reliable operation. It will beappreciated by those skilled in the art, that even with an anodefabricated in accordance with the present disclosure, replacement of theanode, which preferably comprises the inner electrode 128 represented inFIG. 15A, may be necessary after a period of use. In some embodimentsthe construction facilitates replacement of the inner electrode 128 andthe outer electrode 126 when it becomes necessary.

Represented in FIG. 16 is a block diagram of a second presentlypreferred embodiment, generally represented at 150. The embodimentrepresented in FIG. 16 is particularly adapted for treating largequantities of saline solution. Represented in FIG. 16 is a tank 152 inwhich the saline solution is electrolyzed. An electrode assembly 154 isprovided in the tank and is preferably immersed into the solution. Apower supply 158, capable of providing sufficient current at the propervoltage, is connected to the electrode assembly via a cable 160.

Also represented in FIG. 16 is a circulation device 156 which optionallyfunctions to circulate the solution within the tank 152. A sensor 162 isalso optionally provided to measure the progress of the electrolyzationof the solution in the tank 152, for example by measuring the pH of thesolution. The sensor may preferably be an ion selective electrode whichcan be chosen from those available in the art. Other sensors, forexample chlorine, ozone, and temperature sensors, may also be included.A control unit 164 is optionally provided to coordinate the operation ofthe power supply 158, the circulation device 156, and the sensor 162 inorder to obtain the most efficient operation of the apparatus 150.

It will be appreciated that devices such as power supply 158,circulation device 158, sensor 162, and control unit 164 can be readilyobtained from sources in the industry and adapted for use with someembodiments by those skilled in the art using the information containedherein. In particular, the control unit 164 is preferably a digitalmicroprocessor based device accompanied by appropriate interfaces allallowing for accurate control of the operation of the apparatus 150.Some embodiments also can include structures to prevent contamination ofthe treated solution by contact with nonsterile surfaces and by airbornepathogens both during treatment and while the fluid is being transferredto the apparatus and being withdrawn from the apparatus.

Reference will next be made to FIGS. 17 and 18 which are a top view andcross sectional view, respectively, of an electrode assembly, generallyrepresented at 154, which is preferred for use in the apparatusrepresented in FIG. 16. As can be seen best in FIG. 17, the electrodeassembly 154 includes a plurality of concentrically arranged anodes andcathodes. The cylindrical shape and concentric arrangement of theelectrodes represented in FIG. 17 provides for the most efficientoperation. The number of electrodes which are included can be selectedaccording to the application of the apparatus. For example, the numberof electrodes may be six, seven, eight, the eleven represented in FIGS.17 and 18, or more.

In FIG. 17, electrodes 170, 174, 178, 182, 186, and 190 preferablyfunction as cathodes and are preferably fabricated in accordance withthe principles set forth above in connection with the outer electroderepresented at 126 in FIGS. 14-1SA. Furthermore, in FIG. 17 electrodes172, 176, 180, 184, and 188 function as anodes and are preferablyfabricated in accordance with the principles set forth above inconnection with the inner electrode represented at 128 in FIGS. 14-15A.

In the cross sectional side view of FIG. 18 a plurality of tabs extendfrom the cylindrical electrodes 170, 172, 174, 176, 178, 180, 182, 184,186, and 190 to facilitate making an electrical connection thereto.Provided below in the following Table are the relationship between thetabs illustrated in FIG. 18 and the electrodes.

Relationship between the tabs illustrated in FIG. 18 Electrode TabFunction 170 170A Cathode 172 172A Anode 174 174A Cathode 176 176A Anode178 178A Cathode 180 180A Anode (Not illustrated in FIG. 18) 182 182ACathode 184 184A Anode 186 186A Cathode 188 188A Anode 190 190A Cathode(Not illustrated in FIG. 18)

Using the tabs 170A, 172A, 174A, 176A, 178A, 180A, 182A, 184A, 186A,188A, and 190A, those skilled in the art can provide the necessaryelectrical connections to the electrodes 170, 172, 174, 176, 178, 180,182, 184, 186, and 190 and can also provide numerous structures toprevent contact between the tabs and the fluid to be treated. Each ofthe tabs illustrated in FIG. 18 are provided with an aperture, such asthose represented at 1728, 1768, and 1848, which receive a wiringconnector.

While the apparatus described in Example 3 herein has many uses, themost preferred use of the apparatus described herein is subjectingsterile saline solution to electrolysis. The electrolyzed salinesolution can then be used to treat a patient. The saline solutionpreferably has an initial concentration in the range from about 0.25% toabout 1.0% NaCl which is about one-fourth to full strength of normal orisotonic saline solution. According to Taber's Cyclopedic MedicalDictionary, E. A. Davis, Co. 1985 Ed., an “isotonic saline” is definedas a 0.16 M NaCl solution or one containing approximately 0.95% NaCl; a“physiological salt solution” is defined as a sterile solutioncontaining 0.85% NaCl and is considered isotonic to body fluids and a“normal saline solution;” a 0.9% NaCl solution which is consideredisotonic to the body. Therefore, the terms “isotonic,” “normal saline,”“balanced saline,” or “physiological fluid” are considered to be asaline solution containing in the range from about 0.85% to about 0.95%NaCl. Moreover, in accordance with some embodiments, a saline solutionmay be subjected to electrolysis at concentrations in the range fromabout 0.15% to about 1.0%.

It is preferred that one of the above described saline solutions bediluted with sterile distilled water to the desired concentration,preferably in the range from about 0.15% to about 0.35% prior totreatment. This dilute saline solution is subjected to electrolysisusing some embodiments of the present disclosure at a voltage, current,and time to produce an appropriately electrolyzed solution as will bedescribed shortly. It is presently preferred to carry out theelectrolysis reaction at ambient temperatures. In a more preferredembodiment the saline solution used with the apparatus of Example 3 is9.1 g NaCl/1 L of water. In another preferred embodiment the salinesolution used with the apparatus of Example 3 is 2.8 g NaCl/1 L ofwater.

The voltage and current values provided herein are merely exemplary andthe voltage and current values which are used, and the time the salinesolution is subject to electrolysis, is determined by many variables,e.g., the surface area and efficiency of the particular electrodeassembly and the volume and/or concentration of saline solution beingelectrolyzed. For electrode assemblies having a different surface area,greater volumes of saline solution, or higher concentrations of salinesolutions the voltage, current, or time may be higher and/or longer thanthose exemplary values provided herein. In some embodiments, it is thegeneration of the desired concentration of ozone and active chlorinespecies which is important. Electrolyzation of the saline solution alsoresults in other products of the electrolysis reaction including membersselected from the group consisting of hydrogen, sodium and hydroxideions. It will be appreciated that the interaction of the electrolysisproducts results in a solution containing bioactive atoms, radicals orions selected from the group consisting of chlorine, ozone, hydroxide,hypochlorous acid, hypochlorite, peroxide, oxygen and perhaps othersalong with corresponding amounts of molecular hydrogen and sodium andhydrogen ions.

In order to arrive at the preferred end product, electrolyzed salinesolution using the apparatus illustrated in FIGS. 14-15A, about a 0.33%(about one third physiologically normal) saline solution is placed inthe fluid vessel 116 (FIG. 14) and the apparatus is operated for about 5to 15 minutes with a voltage between the electrodes being maintained inthe range from about 10 volts to about 20 volts with a current flowmaintained in the range from about 5 to about 20 amps. In one example,the cell described in Example 3 operated for 1 hour at 40 C using 3 Ampswith a saline solution of less than 0.35% saline. In one example, thecell described in Example 3 operated for 1 hour at 40 C using 3 Ampswith a saline solution of less than 1.0% saline. In one example, thecell described in Example 3 operated for 3 minutes at 23 C using 3 Ampswith a saline solution of less than 0.35% saline. In one example, thecell described in Example 3 operated for 3 minutes at 23 C using 3 Ampswith a saline solution of less than 1.0% saline.

As one example of the use of the embodiment of FIGS. 14-15A, a 0.225%saline solution is subjected to a current of 3 amperes at 20 volts (DC)for a period of three minutes. A 17 ml portion of this electrolyzedsolution is aseptically diluted with 3 ml of a sterile 5% salineresulting in a finished isotonic electrolyzed saline having an activeozone content of 12+/−2 mg/L and an active chlorine species content of60+/−4 ppm at a pH of 7.4.

In some embodiments, low voltages can be used, preferably not greaterthan forty (40) volts DC or an equivalent value if other than directcurrent is used. More preferably, in some embodiments the voltages usedare not more than about thirty (30) volts DC. The use of low voltagesavoids the problem of production of undesirable products in the fluidwhich can result when higher voltages are used. In some embodiments, theclose spacing of the electrodes facilitates the use of low voltages.

In another example, to show that the embodiment of FIGS. 14-15 can beused to effectively carry out electrolysis in saline solutions up toabout 1% in concentration, the electrolysis reaction is carried out atsaline concentrations of 0.3, 0.6 and 0.9%, respectively. The activechlorine species Cl₂ and ozone O₃ contents were measured and areprovided in the table below:

Cl₂ and O₃ Content from Salines at Varying Concentrations SalineConcentration Cl₂ Concentration O₃ Concentration (% NaCl) (ppm) (mg/ml)0.3 129 21.8 0.6 161 26.6 0.9 168 28.0

As can be seen from the above table, the resulting electrolyzed salinesolution includes active components which are within the parametersrequired for effective treatment. [000281]

It will be appreciated that the features of the present disclosure,including the close electrode spacing, the low voltages used, and thematerials used to fabricate the electrodes, result in an apparatus whichprovides unexpectedly better results than the previously availabledevices and schemes.

Example 4

A saline solution was made with the apparatus of Example 3 wherein thesolution was electrolyzed for 3 min at 3 amps and such that the solutionbeing electrolyzed had 9.1 g NaCl/L of purified water. The product madeaccordingly is called RXN-1. The RXN-1 product was tested forsuperoxides and hypochlorites as described herein. Specifically, thepresence of superoxides was tested with the Nanodrop 3300 andR-phycoerytherin (R-PE) as the reagent and the presence of hypochloriteswas tested with the Nanodrop 3300 and aminophenyl fluorescein (APF) asthe reagent. The tests revealed the presence of both superoxides as wellas hypochlorites. The superoxides were tested as an amount relative tothe amount of superoxides that are present in a sample made according toExample 1. That is, superoxides were tested as an amount relative to theamount of superoxides when a total of 1,000 gallons of salinated waterwas electrolyzed with a total of 56 amps running through the electrodesand further wherein the electrolyzing occurred at 4.5-5.8° C. The amountof superoxides present in the RXN-1 product was 130% of the amount ofsuperoxides present in a sample made according to Example 1. Similarly,the hypochlorites were tested as an amount relative to the amount ofhypochlorites that are present in a sample made according to Example 1.That is, hypochlorites were tested as an amount relative to the amountof hypochlorites when a total of 1,000 gallons of salinated water waselectrolyzed with a total of 56 amps running through the electrodes andfurther wherein the electrolyzing occurred at 4.5-5.8° C. The amount ofhypochlorites present in the RXN-1 product was 82% of the amount ofhypochlorites present in a sample made according to Example 1.

Example 5

A saline solution was made with the apparatus of Example 3 wherein thesolution was electrolyzed for 3 min at 3 amps and such that the solutionbeing electrolyzed had 2.8 g NaCl/L of purified water. The product madeaccordingly is called RXN-2. The RXN-2 product was tested forsuperoxides and hypochlorites as described herein. Specifically, thepresence of superoxides was tested with the Nanodrop 3300 andR-phycoerytherin (R-PE) as the reagent and the presence of hypochloriteswas tested with the Nanodrop 3300 and aminophenyl fluorescein (APF) asthe reagent. The tests revealed the presence of both superoxides as wellas hypochlorites. The superoxides were tested as an amount relative tothe amount of superoxides that are present in a sample made according toExample 1. That is, superoxides were tested as an amount relative to theamount of superoxides when a total of 1,000 gallons of salinated waterwas electrolyzed with a total of 56 amps running through the electrodesand further wherein the electrolyzing occurred at 4.5-5.8° C. The amountof superoxides present in the RXN-2 product was 120% of the amount ofsuperoxides present in a sample made according to Example 1. Similarly,the hypochlorites were tested as an amount relative to the amount ofhypochlorites that are present in a sample made according to Example 1.That is, hypochlorites were tested as an amount relative to the amountof hypochlorites when a total of 1,000 gallons of salinated water waselectrolyzed with a total of 56 amps running through the electrodes andfurther wherein the electrolyzing occurred at 4.5-5.8° C. The amount ofhypochlorites present in the RXN-2 product was 80% of the amount ofhypochlorites present in a sample made according to Example 1.

Power Sources

As described in detail above, a DC (direct current) is used toelectrolyze water.

To prepare a direct current for electrolyzation, readily availableelectricity, such as that which comes from a wall socket, is brought toa terminal strip. This terminal strip, also known as a terminal block,acts like a surge protector allowing a number of electrical connectionsfrom the strip to other devices. For example, the terminal strip can bean interface for electrical circuits. The terminal strip can beconnected to a ground and/or a current transformer. A transformer can beused to measure electric currents. The terminal strip can also beconnected to a potentiometer. The potentiometer measures voltage acrossan electrical system and can be used to aid in adjusting the voltage.For example a dial can be connected to the potentiometer so that theoperator may adjust the voltage as desired.

Another transformer can be connected to the potentiometer, which canthen be operably connected to a rectifier. Rectifiers in general convertalternating current (AC) to direct current (DC). One specific type ofrectifier which can be used is a bridge rectifier. Converting thewaveform into one with a constant polarity increases the voltage output.This waveform is called a full wave rectified signal. Once the waveformand voltage are configured as desired, the DC shunt can provide a meansfor bringing electricity to different devices such as the electrodes,monitors and other operational instruments.

FIG. 19 diagrams an example of a power source which can be used in someembodiments. Electricity comes in from the wall 10 and is met by aterminal strip 11. Terminal strip 11 is in operable communication with apotentiometer 12, and a current transformer 13. Potentiometer 12 is inoperable communication with the transformer 13. The transformer 13 is inoperable communication with a rectifier 14.

FIG. 20 diagrams an example of a power source which can be used in someembodiments. Electricity comes in from the wall 102 and is met by aterminal strip 103. Terminal strip 103 is in operable communication witha potentiometer 105, a grounding means 101 and a current transformer104. Potentiometer 105 is in operable communication with the transformer106. The transformer 106 is in operable communication with a rectifier107. Rectifier 107 is in operable communication with a DC shunt 108.

Determination of ROS Levels Against a Known Standard

The measurement of concentrations of ROS, particularly a superoxide,inside the solutions has been done by means of a fluoro spectrometer,Nanodrop 3300, and three varieties of fluorescent dyes, R-Phycoerytherin(R-PE), Hydroxyphenyl fluorescein (HPF) and Aminophenyl fluorescein(APF), that are commonly used to determine relative ROS concentrationsinside active biological systems and cells. The molecules in these dyeschange shape, and therefore fluoresce only when exposed to molecularcomponents in ROS. The resulting change in fluorescence can then bedetected by the fluoro spectrometer and can be related to theconcentration of ROS present. ROS concentrations in electrolyzed salinesolutions (ESS) solutions are verified and detected by either APF orR-PE fluorescent dyes, both of which produce entirely consistentmeasurements of relative concentrations of ROS in various concentrationsand dilutions of ESS solutions. ROS measurements in ESS solutions havebeen linked using R-PE fluorescent dye, to the reaction of this dye toregulated concentrations of2/2′-Axobis(2-methylpropionamide)dihidrochloride, a molecule thatproduces known amounts of ROS. This is not an absolute measurement, butit relates ROS in ESS to amounts of a known producer of ROS.

These fluorescent dyes are often used in combination with a fluorescencemicroscope to create high-resolution images of the build-up of ROS(oxidative stress) inside individual living cells. These dyes have beenshown to specifically be sensitive to concentrations of ROS regardlessof complex surrounding chemical environments.

Although APF and R-PE dyes are capable of measuring relative ROSconcentrations in ESS solutions, no known absolute standardconcentration for stabilized ROS in pure saline solutions exists.Furthermore, discrepancies in the decay time of these fluorescent dyesmake measuring standardized amounts of ROS in other solutionsincompatible with measuring those found in ESS. This may be due, inpart, to the molecular complexes in ESS solutions that keep the ROSconcentration stable, effectively shielding the free radicals fromreadily reacting with the dyes. The standard for ROS concentration inESS solutions is therefore measured relative to the ROS concentration ina standardized solution that has been used in all of the antimicrobialand toxicity studies to date, both published and unpublished. Methods tomeasure absolute ROS concentrations in ESS solutions are actively beingpursued.

The regulated amounts of ROS, thus measured, inside a variety of the ESSsolutions produced in some embodiments have been shown to be stable,consistent and predictable, sufficient for therapeutic applications.

The development of a phycobiliprotein fluorescence quenching assay forthe routine determination of ROS content in ASEA has been successful andis used routinely to monitor production quality for ROS levels. Theassay has the following characteristics: ease of use, sensitivity, andquantitation. The assay is linear over a 2 log 10 range of ROSconcentrations. For a compositions comprising RXNs, the starting salinewas used as a negative control, AAPH (2,2′-Azobis (2-amidinopropane)dihydrochloride which is a standard ROS generating compound) served as apositive control and allowed the generation of a standard curve, and thecompositions comprising RXNs or other samples comprised the unknowns.

For the purposes of this work, we determined the oxygen radical contentof our health benefiting product. In the assay described below,R-Phycoerythrin [an algal protein] is exposed to varying levels of astandard ROS generating compound [AAPH] wherein the level offluorescence quenching is logarithmically related to ROS content. Thisprovides a standard curve from which to estimate the ROS content ofunknown samples. The levels of ROS in the so unknown samples areexpressed as mM equivalents of AAPH. FIG. 24 shows the concentration ofAAPH.

Materials and Methods:

PHYCOERYTHRIN and R-PHYCOERYTHRIN: were purchased from SigmaChemicalCorporation, St. Louis, Mo. AAPH: 2,2′-azobis (2-amidino-propane)dihydrochloride was purchased from Wako Chemicals USA, Richmond, Va.This compound generates ROS upon contact with water.

FLUORESCENCE READER: an 8 or 16 place fluorescence reader manufacturedby Pacific Technologies, Redmond, Wash. was used to detect thefluorescence signal from the phycoerythrins. Temperature was controlledat 37 C during a 12-20 hr. experimental run. The samples wereinterrogated every 0.5 to 2 min where each sample interrogation wascomprised of 1024 lamp flashes from a LED whose emission spectra wasappropriate from the excitation spectra of R-Phycoerythrin. Propercut-off filters were employed to detect the fluorescence emissions ofthe phycoerythrins.

DATA ANALYSES: All data is captured in real time. The data contained inthe worksheet can be manipulated to determine the relative change offluorescence over the time course of the experiment and subsequently,SigmaPlot Pro v. 7 software [SPSS Software, Chicago, Ill.] is used todetermine the area under the curve. Area under the curve [AUC] analysisis appropriate since Cao, Cao et al. Comparison of different analyticalmethods for assessing total antioxidant capacity of human serum.Clinical Chemistry June 1998 vol. 44 no. 6 1309-1315 which is herebyincorporated by reference in its entirety, and colleagues havedemonstrated that in this method both the inhibition time and degree ofinhibition of fluorescence by free radicals are considered. The areaunder the curve [AUC] are plotted against the log 10 mM AAPHconcentration to provide a standard curve from which to estimate thelevels of ROS in unknown samples.

Detailed Methods:

Step a. 300 uL of phosphate buffer, pH 7.0, 100 mM is added to W′ glassvials. Step b. 15 ug of R-Phycoerythrin in 15 uL of phosphate buffer isadded to the materials in Step a. The vials are capped and placed intothe wells of the fluorescence reader for 15 min prior to the addition ofa saline control, ASEA or AAPH solutions. During this period,fluorescence values are collected from which to calculate a 100% value.This value is then used in subsequent calculations to determine arelative fluorescence signal value for the standard curves.

One mg of AAPH is added to 1 ml of phosphate buffer and 10-folddilutions are made to provide at least a 3 log 10 range of AAPHconcentrations. Similarly, ASEA solutions are diluted and added toappropriate vials in Step b.

100 ul of the materials in Step a are added to the appropriate vials inStep b. The vials are mixed and replaced into the reader for up to anadditional 12 to 20 hrs of evaluation.

RESULTS: As shown in FIG. 24, as the concentration of AAPH decreasedfrom 1.00 mM to 0.050 mM, there was as concomitant increase in thenormalized AUC. Buffer control [not shown] revealed that over time thereis a spontaneous loss of fluorescence signal, although this lossrepresents only−8% of the original signal.

The data represented in FIG. 25 shows intra-assay variability of twoconcentrations of AAPH. Using SigmaStat v 2.01 software, the followingmean, Std Deviation and Relative Std Deviation were calculated and arepresented in Table 1. The data shows that the variation for eachconcentration the variation among replicates ranged from −0.1% to 4%variation [Rel. Std. Dev.]. These data suggest that fluorescencequenching assay is capable of producing small variations amongtriplicate or quadruplicate samples over a 10-fold range of AAPHconcentrations.

TABLE 1 Intra-Assay Variability AUC Values AAPH % Rel. Concentration NMean AUC Std. Dev. Std. Error Std. Dev.  3.69 mM 3 653 1.07 0.62 0.150.369 mM 4 804 31.7 15.0 3.7

Table 2 shows the results of the analyses of ASEA solutions prepared byMDI and filtered through 0.2 μm Supor membrane to ensure sterility priorto clinical application. It is clear that the ASEA from differentproduction lots are similar in their ROS content. Statistical analysissupported this observation [p=0.272]. The most important point is theobservation that filtration through a 0.2 μm Supor membrane does notdecrease the ROS content of ASEA.

TABLE 2 ROS Content of ASEA Filtered and Unfiltered Through 0.2 μm SuporMembrane Treatment N Mean AUG Std. Dev. Std. Error % Rel. Std. Dev.Unfiltered 4 589.7 65.8 32.9 5.5 Filtered 4 646.3 66.3 33.1 5.1

The levels of variance [Rel. Std. Dev.] reported here is similar to thatreported by Cao and colleagues.

In Table 3, data from a typical analysis is illustrated. Saline[negative control] always contained less than 0.1 mM AAPH equivalents ofROS whereas ASEA always contained >1.0 mM ROS.

TABLE 3 ROS Content of ASEA and Saline ASEA or Saline ROS Content mMSamples Mean AUG AAPH equivalents ASEA 479 3.3 ASEA 543 2.2 ASEA 441 4.5ASEA 523 2.98 ASEA 516 3.2 Saline 974 0.095 Saline 956 0.075

The above shows a known concentration of a standard, AAPH, as 653 and804 when tested at 3.69 mM and 0.369 mM respectively. A compositioncomprising RXNs showed an AUC of between 441-543.

The measurement of concentrations of ROS inside the solutions can bedone by means of a fluorospectrometer, Nanodrop 3300, and threevarieties of fluorescent dyes, R-Phycoerytherin (R-PE), Hydroxyphenylfluorescein (HPF) and Aminophenyl fluorescein (APF), all of which arecommonly used to determine relative ROS concentrations inside activebiological systems and cells. The molecules in these dyes change shape,and therefore fluoresce only when exposed to molecular components inROS. The resulting change in fluorescence can then be detected by thefluorospectrometer and can be related to the concentration of ROSpresent. ROS concentrations in compositions comprising RXNs can beverified and detected by either APF or R-PE fluorescent dyes, both ofwhich produce entirely consistent measurements of relativeconcentrations of ROS in various concentrations and dilutions of RXNs.The ROS measurements in a compositions comprising RXNs have been linked,using R-PE fluorescent dye, to the reaction of this dye to regulatedconcentrations of 2/2′-Axobis(2-methylpropionamide) dihidrochloride, amolecule that produces known amounts of ROS.

Superoxide Testing

Superoxides were tested with the NanoDrop 3300 and R-PE as the reagentfor the three samples.

The intensity of the fluorescence indicates the amount of ROS in thesample. This dye, R-PE, is toxic, expensive, must be kept refrigerated,degrades in strong blue light, such as a fluorescent bulb, and is timesensitive. The following steps were taken:

The ND-3300 software was called up, the “Other Fluorophores” button wasclicked and the “R-PE SOuM Activated” option was selected.

The ND-3300 was blanked: 2 ul (1 drop) of deionized water was placedusing a pipette on the measurement pedestal and the arm was carefullyclosed. The “Blank” button was clicked and the ND-3300 took a “blank”measurement, thereby calibrating the ND-3300.

The samples were prepared by pipetting 10 ml deionized water into eachone of the large (15 ml) test tubes required for the test. One test tubewill be required for each sample to be tested.

The test tubes were labeled by cutting out squares of sticky-back labelstock, large enough to fit over the mouth of the test tubes, and bywriting the number “1”, “2” and “3” on the label. The labels were placedcovering the mouth of the test tubes to both identify them and to keepthe liquids from evaporating.

10 μl of the R-PE fluorescent dye was apportioned into each of the testtubes by following these steps: turning off the lights, taking thepreviously prepared R-PE dye test tube out of the refrigerator [thistest tube was previously prepared by putting 2 μl of the concentratefrom the commercial R-PE vial inside 5 ml deionized water (a phosphatebuffer is not needed)]. The prepared test tube was placed in the rackwith the others. This dye is toxic and is sensitive to light so thesesteps should be done quickly, with lab coat, gloves and goggles. With aclean pipette, 10 μl of the prepared R-PE dye was add into each of thetest tubes. The R-PE was placed back in the test tube back in therefrigerator.

The test tubes were mixed well using a mixing pipette which was placeinto each of the test tubes, 2-3 ml were drawn out and then quicklypushed back in, allowing some bubbles to escape to better agitate thecontents of the test tubes. This was repeated three to four times foreach tube. At this point, it is necessary to have separate mixingpipette heads for each tube. The test tubes were allowed to sit forleast 30 min. after mixing.

The initial pre-sample measurements were taken on all of the test tubes:The ND-3300 was blanked using the procedures outlined above. A foldedKimwipe was used to blot the last sample droplet off the lower and upperpedestals before loading a new drop to be analyzed. A descriptive namefor the sample was typed into the Sample ID field in the software.

2 μl of test tube #1 was loaded onto the pedestal, the arm was carefullyclosed and the “measure” button pressed. Three measurements were takenof the sample in test tube #1. This procedure was repeated for the nexttwo samples. Specifically, the Sample ID field was changed to reflectthe descriptive name of the sample in the second test tube. And thenthree (3) measurements were taken from the second test tube also. Thisstep was done until all test tubes were analyzed. When R-PE wasactivated, the RFU readings shown were between the 100 and 2000.

A composition comprising RXNs was added to the test tubes: Thisprocedure was carefully timed. The R-PE dye is only accurate for lessthan 30 minutes after activation and therefore all measurements must beacquired after the same amount of exposure time. 10 μl of a compositionscomprising RXNs sample #1 was added to test tube #1 and immediatelythereafter a timer was set for three (3) minutes. Then the test tube #1was mixed with a pipette. This step was repeated for all three samples.

At 6 h post addition of the first a compositions comprising RXNs sampleto a test tube, measurements were taken from every test tube in thefollowing manner. The ND-3300 was blanked, the pedestals were blottedand the “Sample ID” for test tube #1 was typed in. After three (3)minutes, using a sampling pipette, a 2 μl drop was taken from test tube#1 and place it on the pedestal and the measure button was pressed. Thisprocess was repeated until all of the test tubes were measured.

The data was cleaned up by pressing the “Show Report” button so that allof the data that has been taken so far was displayed. The data was thensaved and analyzed.

Hypochlorite Testing

Hypochlorites were tested with the NanoDrop 3300 Fluorospectrometer andAPF as the reagent.

The ND-3300 software was called up, the “Other Fluorophores” button wasclicked and the “APF 50 uM Activated” option was selected.

The ND-3300 was blanked: 2ul (1 drop) of deionized water was placedusing a pipette on the measurement pedestal and the arm was carefullyclosed. The “Blank” button was clicked and the ND-3300 took a “blank”measurement, thereby calibrating the ND-3300.

The samples were prepared by pipetting 10 ml deionized water into eachone of the large (15 ml) test tubes required for the test. One test tubewill be required for each sample to be tested.

The test tubes were labeled by cutting out squares of sticky-back labelstock, large enough to fit over the mouth of the test tubes, and bywriting the number “1”, “2” and “3” on the label. The labels were placedcovering the mouth of the test tubes to both identify them and to keepthe liquids from evaporating.

10 μl of the APF fluorescent dye was apportioned into each of the testtubes by following these steps: turning off the lights, taking thepreviously prepared APF dye test tube out of the refrigerator [this testtube was previously prepared by putting 2 μl of the concentrate from thecommercial APF vial inside 5 ml deionized water (a phosphate buffer isnot needed)]. The prepared test tube was placed in the rack with theothers. This dye is toxic and is sensitive to light so these stepsshould be done quickly, with lab coat, gloves and goggles. With a cleanpipette, 10 μl of the prepared APF dye was add into each of the testtubes. The APF was placed back in the test tube back in therefrigerator.

The test tubes were mixed well using a mixing pipette which was placeinto each of the test tubes, 2-3 ml were drawn out and then quicklypushed back in, allowing some bubbles to escape to better agitate thecontents of the test tubes. This was repeated three to four times foreach tube. At this point, it is necessary to have separate mixingpipette heads for each tube. The test tubes were allowed to sit forleast 30 min. after mixing.

The initial pre-sample measurements were taken on all of the test tubes:The ND-3300 was blanked using the procedures outlined above. A foldedKimwipe was used to blot the last sample droplet off the lower and upperpedestals before loading a new drop to be analyzed. A descriptive namefor the sample was typed into the Sample ID field in the software.

2 μl of test tube #1 was loaded onto the pedestal, the arm was carefullyclosed and the “measure” button pressed. Three measurements were takenof the sample in test tube #1. This procedure was repeated for the nexttwo samples. Specifically, the Sample ID field was changed to reflectthe descriptive name of the sample in the second test tube. And thenthree (3) measurements were taken from the second test tube also. Thisstep was done until all test tubes were analyzed. When APF wasactivated, the RFU readings shown were between the 100 and 2000.

A composition comprising RXNs was added to the test tubes: Thisprocedure was carefully timed. The APF dye is only accurate for lessthan 30 minutes after activation and therefore all measurements must beacquired after the same amount of exposure time. 10 μl of a compositionscomprising RXNs sample #1 was added to test tube #1 and immediatelythereafter a timer was set for three (3) minutes. Then the test tube #1was mixed with a pipette. This step was repeated for all three samples.

APF, as well as RPE, are measured relative to a chosen standard and arereported as percentages of such standard.

At 30 min. post addition of the first a compositions comprising RXNssample to a test tube, measurements were taken from every test tube inthe following manner. The ND-3300 was blanked, the pedestals wereblotted and the “Sample ID” for test tube #1 was typed in. After three(3) minutes, using a sampling pipette, a 2 μl drop was taken from testtube #1 and place it on the pedestal and the measure button was pressed.This process was repeated until all of the test tubes were measured.

Packaging

The packaging process includes any type of packaging that does notcontribute to the decay of the superoxides, hydroxyl radicals and OOH*(for example, containers should not contain metal oxides or ions).Pouches and bottles are preferred for ease of portability andacceptability in the market. However, any suitable packaging isapplicable. Containers/packaging can be made of for example glass,polyethylene, polypropylene and the like. Specific examples includeBapolene HD2035, which is a high density polyethylene copolymer and Jadebrand CZ-302 polyester. Table 4 shows the relative percentage ofsuperoxides remaining after a 12 month period when the composition ispackaged in a polyethylene bottle.

Example 6

The rate of decay for superoxides, from a sample made according toExample 1, was tested over a 12 month period. That is, superoxidespresent in a sample made when a total of 1,000 gallons of salinatedwater is electrolyzed with a total of 56 amps running through theelectrodes and further wherein the electrolyzing occurred at 4.5-5.8°C., according to Example 1, were tested for their relative amounts overa period of 12 months relative to a standard RFU control for RPE.

TABLE 4 1 Year Studies—shows a 3%/month decay rate over a 12 monthperiod % Potency/ Stability as RFU compared Average RFU to per minusStandard reference Sample ID RFU sample control deviation % error sampleRFU Control 1743.7 1759.033 Control 1814.6 Control 1718.8 Sample 1 985.6986.1667 872.8667 6.169549 0.706815 1 Sample 1 980.3 Sample 1 992.6Sample 2 1044.8 1003.6 855.4333 35.68151 4.171162 Baseline Sample 2982.7 Sample 2 983.3 Sample 3 981.7 988.3 870.7333 16.23915 1.8649971.007618 Sample 3 1006.8 Sample 3 976.4 Sample 4 1132.9 1121.133 737.912.56437 1.70272 0.853903 Sample 4 1107.9 Sample 4 1122.6 Sample 51189.9 1182.2 676.8333 19.99475 2.954161 0.783236 Sample 5 1197.2 Sample5 1159.5 Sample 6 1269.3 1256.267 602.7667 26.47647 4.39249 0.697526Sample 6 1225.8 Sample 6 1273.7

Table 4 provides data for the RFU control, Sample 1 which is a referencesample and Samples 2-6 which were taken at 1 month, 3 months, 6 monthsand 12 months respectively. Table 4A shows the results as a percentageof remaining superoxides at 0, 1, 3, 6 and 12 months.

This Table 4 is graphically represented in FIG. 22.

TABLE 4A Month % Potency/Stability 0 100 1 101 3 85 6 78 12 70

Example 7

Table 5 shows the relative percentage of superoxides remaining after a13 month period when the composition is packaged in a polyethylenebottle and polyethylene pouch. In this Example, the composition testedwas made according to the process of Example 6.

TABLE 5 13 Month Pouch v. Bottle % Potency/ RFU Stability as Average RFUcompared to Sample per Standard minus reference ID RFU sample deviation% error control sample Control 1687.9 555 946.4 940.7667 9.1576930.973429 1325.273 1 555 930.2 1370.007 555 945.7 555-1 817.5 851.329.27781 3.439188 1414.74 1.067508 555-1 867.6 555-1 868.8 525b 967.2966.0333 10.3992 1.076484 1300.007 0.948905 525b 955.1 525b 975.8 524p983.1 975.7333 17.08576 1.751069 1290.307 0.941825 524p 956.2 524p 987.9480 985.9 1006.333 19.12337 1.900302 1259.707 0.919489 480 1009.3 4801023.8 479p 1115.2 1153.5 45.22975 3.921088 1112.54 0.812069 479p 1141.9479p 1203.4 408p 1454.2 1501.633 62.98812 4.194641 764.4067 0.557958408p 1573.1 408p 1477.6 347p 1309.4 1327.833 39.24364 2.955464 938.20670.684819 347p 1301.2 347p 1372.9 347p 1338.1 314 1354.4 1348.56716.82627 1.247715 917.4733 0.669685 314 1361.7 314 1329.6 313p 1459.31444.033 13.25908 0.918198 822.0067 0.600002 313p 1435.4 313p 1437.4

The above graph shows a 4.4% decay rate of the superoxide radical forthe pouch and a 3% decay rate for the bottle over a 13 month period.Sample 555 is a reference sample, Sample 555-1 is a baseline sample,Sample 525b is a sample taken from a bottle after 1 month, Sample 524pis a sample taken from a pouch after 1 month, Sample 480 is a Sampletaken from a bottle after 3 months, Sample 479p is a sample taken from apouch after 3 months, Sample 408p is a sample taken from a pouch after 8months, Sample 374p is a sample taken from a pouch after 11 months,Sample 314 is a sample taken from a bottle after 13 months and Sample313p is a sample taken from a pouch after 13 months. Table 5A is a chartshowing the percentage of remaining superoxides at 0, 1, 3, 8, 11 and 13months in a bottle and a pouch type container. This Table 5 isgraphically represented in FIG. 23.

TABLE 5A Bottle % Pouch % Month Potency/Stability Potency/Stability 0100 100 1 95 94 3 92 81 8 56 11 68 13 67 60

Example 8

Borosilicate glass, such as those sold under the trade names of Kimax,Pyrex, Endural, Schott, or Refmex for example, are useful for packagingof compositions comprising RXNs.

The presence of superoxides in compositions comprising RXNs samples weretested after being stored in borosilicate glass bottles. The sampleswere made according to the process described in Example 6. Sample 397had been stored for 24 months and Sample 512 had been stored for 20months. Reference batch 1256 was made the same day as the test was runon all three samples. The Results are shown in Table 6.

TABLE 6 Glass Bottle ASEA Stability % Potency/ Control − Stability asaverage average + compared to Sample RFU RFU control loss referencesample 397 780.5 806.8 1193.2 93.1169 819.5 820.4 512 676.7 682.46666671317.533333 102.8198 682.6 688.1 Reference 754.8 718.6 1281.4 100 sample1256 707.2 693.8 Control 1850 Control after 1700 6 hours

It can be seen from the Tables that the relative concentrations ofsuperoxides do not appreciably degrade while in the borosilicatebottles. Sample 397 had a decayed about 5% and sample 512 had 0% decay.Therefore, the yearly decay of product is no more than about 2.5% decayper year. This gives an estimated half-life of the superoxides at about24 years.

The stability of any component in the composition can be measured by theamount of the particular composition which remains detectable after acertain amount of time. For example, if the superoxides measured had adecay rate of about 7% over a two year period, this would mean that thestability over the 2 year period was about 93%. In other words, after atwo year period, about 93% of the original amount of superoxides, werestill present and measured in the composition.

Example 9

Two 1 L 0.9% NaCl solutions were made and three 0.28% 1 L NaCl solutionswere made from 0.28% distilled NaCl solutions. Salinity was analyzedwith an EC300 conductivity meter and salt was added until the desiredsalinity (9 g/L or 0.9%) was reached. Samples were then mixed and placedin the freezer. 0.28% samples were collected directly from the salinestorage tanks. Salinity was confirmed at 2.8 g/L (or 0.28%) by the EC300conductivity meter. Samples were placed in the freezer.

Samples were removed from the freezer when the temperature read at 5.5°C. and placed in the fridge. One of the 2.8 g/L sample was run at 3 ampsfor 3 min at 5.8° C. to rinse the 1 L cell, after which the samples inthe following table were run similar to the process of Example 3.

Sample Salinity (g/L) Amps Time (min) Temp (C.) 1 2.8 g/L 3 3 5.8 2 2.8g/L 3 3 5.8 3  9 g/L 3 3 5.6 4  9 g/L 3 3 4.9

Free Chlorine, R-PE, APF and pH were measured for the 0.28 and 0.9%samples and the results were as shown in the following table.

Sample/NaCl % Free Chlorine R-PE APF pH 1/0.28% 31 ppm 112% 112% 7.63/0.9%  76 ppm 123%  35% 8.3 2/0.28% 112% 108% 4/0.9%  125%  48% *FreeCl was tested using glass cells for 1 in the LR and 3 was measured inplastic cells in the HR.

Example 10

A composition made according to Example 1—KI TITRATION WITH Na₂S₂O₃

A titration was set up to determine the amount of ClO in a compositionmade according to Example 1 (for this Example 10 a composition madeaccording to Example 1 is referred to RXN1) by reacting ClO in RXN 1with KI and acid to make I₂ and CF. The I₂ is brown in color and becomesclear upon complete reaction with S₂O₃— and 2I⁻.

The reagents are KI 42 mM with Glacial acetic acid solution (KIGAA),RXN1 and 0.100 M Na₂S₂O₃ solution. The 42 mM KI solution was prepared byadding 1.758 g of KI and 5 ml of GAA to a 250 ml Erlenmeyer flask andbringing the volume to 250 ml with DI H2O. 0.100M Na₂S₂O₃ solution wascreated by adding 2.482 g of Na₂S₂O₃ to a 100 mL volumetric flask, thenadding DI H₂O until 100 ml was reached. RXN1 was taken from batch 1371.Three tests were performed.

TEST 1: 50 ml of RXN1 was added to 50 ml KIGAA and mixed. The buret wasrinsed three times with DI H₂O then rinsed with Na₂S₂O₃ and filled withNa₂S₂O₃ to 4 ml. Initial buret reading started at 6 ml and ended at 5.69ml. A total of 0.31 ml was added to complete the titration. Resultsindicate about 16 ppm of ClO (3.1×10⁻⁴M ClO).

TEST 2: 75 ml RXN1 was added to a 50 ml KIGAA and allowed to mix.Initial buret reading was 14 ml and final was about 13.55. A total of0.45 ml was added. Results indicate about 16 ppm of ClO (3×10⁴M ClO).

TEST 3: 100 ml RXN1 was added to 50 ml KIGAA. Initial buret reading wasat 15 ml and the final reading was at about 14.37 ml. Approximately 0.63ml was added in total. Results indicate about 16 ppm of ClO (3.15×10⁴MClO).

CONCLUSION: After three Tests it appears that the ClO concentration ofRXN1 is close to 3.1×10⁴M. This corresponds to about 16 ppm which isclose to what the colorimeter read on a sample from another batch (batch1371, which tested at 20 ppm).

Example 11

The AccuTOF-GCv 4G is a highly sensitive (S/N >100 at OFN 1 pg/μL)time-of-flight Gas Chromatography Mass Spectrometer. High resolution andmass accuracy allow for rapid elemental composition determination andtarget compound identification. To test for water clusters in acomposition of some embodiments, the composition was run in the MS andinjection temperatures were lowered to the point where water clusterswere detectable.

The spectra showed the existence of several active oxygen complexes,including ClO— and O2 in complexes with ClO— and the existence of theO2*— radical in several forms. These spectra are shown in FIGS. 26-28.At low mass, we see only water clusters [(H₂O)_(n)+H]+ at 37 and 55,filament temperatures are low enough to not break down water.

Example 12

Hydrogen peroxide was tested by ultravioletvisible (UVNIS) spectroscopyaccording to Standard Test Protocol (STP) Number STP0163 Rev2 by NelsonLaboratories in Salt Lake City. According to this test, hydrogenperoxide was present in a composition at 1.6 ppm by weight.

Example 13

Evaluation and measurements of pH, peroxide, chlorine, free and total,redox and ozone were taken from a composition made according to Example3.

Three initial lots of materials were processed consisting of 15 sub-lotsfor run 1, 30 sub-lots for run 2 and 40 sub-lots for run 3. During run3, sub-lots 1, 15 and 30 were also tested for pH changes and Peroxideproductions as intra-assay sub-lot controls. Starting material was alsotested with each lot to determine which parameters changed duringprocessing. Data showed a change in pH, Peroxide, Chlorine, free andtotal, as well as increased Redox and production of Ozone. There was nochange in osmolarity or chloride levels but a decrease was seen inSodium levels.

Samples from run 3 were also tested after 2 weeks storage at roomtemperature (˜25 C). At this time two samples of the material wereremoved and treated by freeze thaw and by heating to 100 C in order todetermine stability indicating parameters. This data showed that storageat room temperature for 2 weeks changed the Chlorine free and totallevels and ratios from an initial mean value of the three runs of 60 to60 ppm free to total and decreased to 16 to 52 ppm free to total. Freezethawing this material gave values of 36 to 77 ppm, but heating furtherdecreased these values to 8 to 32 ppm. The Sodium values after two weeksstorage also appeared to be lower than the range (1.5 times standarddeviation of the three runs) of 2470 to 4123 ppm down to 2100 ppm. Thishowever did not appear to change (within assay variation) when sampleswere freeze thawed or boiled. The chloride, redox, and peroxide appearedto be within error of the initial data for all three samples (2 week RT,freeze thawed and boiled). Osmolarity was slightly higher for the freezethawed and boiled samples but may be within assay error or was due tothe concentration of the sample caused by treatment.

Prior to initiation of PQ (Performance Qualification) runs, engineeringruns were conducted to determine reproducibility of process and togenerate material for determination of specific testing methods andparameters. Additionally, material was used to determine parameters thatwould be stability indicating. Material was produced using the apparatusand method described in Example 3. Unit has undergone 10/0Q prior tostudy. Sub-lots were prepare using 0.9% sterile injectable Saline at oneliter per sub-lot. Initial run consisted of 15 sub-lots that werepooled, pH adjusted and 0.2 μm filtered. Aliquots were removed forinitial testing using the following Steps.

Steps

1. Visual Inspection: Clear colorless liquid

2. Particulate matter: No visual particles under normal lighting

3. pH: Determination of pH was conducted based on United StatesPharmacopoeia, USP <791> using GBI SOP EC-855. Instrumentation includeda Corning 425 meter and an Accent 13-620-95 combination electrode.System was standardized at 25° C. using NIST traceable buffers that gavea slope of >97%.

4. Osmolarity: Determination of Osmolarity was conducted per USP <785>using an Osmette A model 5002 per GBI SOP AL-872. Unit was standardizedwith NIST traceable calibration standards and a reference control of 290mOsm.

5. Peroxide: Generation of Peroxide was measured using a Peroxide testkit from Merckquant and semi quantitative levels were determined per GBISOP AL-876. This test uses a test strip comparison method to a colorscale. Levels of detection are 0.5, 2, 5, 10 and 25 ppm. Higher-levelsamples can be diluted and measured. Mid color estimates could be doneif necessary.

6. Chlorine total and free: Free Chlorine in the sample as hypochlorousacid or hypochlorite ion (free Chlorine or free available Chlorine)immediately reacts with DPD (N, N-diethyl-p-phenylenediamine) indicatorto form a magenta color which is proportional to the free Chlorineconcentration. Color measurements are made using a Hach Colorimetermodel DR850. Reagent kits are also obtained from Hach. It should be notethat the presence of Ozone interferes with the accurate measurement offree Chlorine and the presence of Peroxides may interfere also.

Chlorine can be present as free or combined available chlorine and ismeasured together as total available chlorine. Combined chlorine exitsas monochloramine, dichloramine, nitrogen trichloride and other chloroderivatives. The combined chlorine oxidizes iodide in the test reagentto iodine. The iodine reacts with DPD along with free chlorine presentin the sample to form a red color that is proportional to the totalchlorine concentration. Combined chlorine can be calculated bysubtracting the free from the total chlorine test result.

It should be noted that ozone and peroxide in the sample might giveinaccurate measurements with these reagents.

7. Redox Potential (ORP): This method measures the oxidizing or reducingcapacity of a solution in mV units. A Platinum Redox Electrode (SympHonyElectrodes) is utilized with a millivolt pH meter. Redox potential isexpressed in terms of a standard electrochemical reduction potential,symbolized as E₀, with millivolt (mV) as units. The value is measuredagainst a standard hydrogen couple (2H⁺, H₂), a universally acceptedframe of reference. By convention, a positive (+) sign accompanies thereduction potential that has a greater tendency to undergo reductionrelative to the hydrogen system. A negative sign is used for solutionsthat have a lesser tendency to undergo reduction. Since the conventionalstandard is pH 7, measurements are pH dependent and appropriatecalculation are required to adjust E₀ value to a condition applicable topH (E o/). Example half-reaction couple potentials for water at 20 to 30C at pH 7 is 820 mV. (O₂+2H₂+2e D H₂O).

8. Chloride: Chloride is measure using a chloride combination electrodefrom Cole-Parmer (27077-04) attached to a IC 7685 Ion controller. Meteris calibrated with a 100 and 1000 ppm chloride standard and samples aremeasured in terms of ppm Cl⁻. A 500 ppm reference standard is also usedto determine reproducibility of the readings for quality purposes.

9. Sodium: Sodium is measure similar to chloride using a sodiumcombination electrode from Cole Parmer (277077-16). Standards of 100 and1000 ppm are used and a 350 ppm reference standard is also used todetermine reproducibility of the readings for quality purposes.

10. Ozone: Measurements of Ozone levels are made using a HACHcolorimeter Indigo method. Method has a detection level of 0.1 ppm.Ozone (O₃) is the gaseous form of oxygen having 3 atoms per moleculerather than the usual 2.

Results: Samples from pre-treated 0.9% Sodium Chloride for injectionwere measured against post treatment product. Table 1 shows the mean,standard deviation (SD) and percent coefficient of variance (% CV) forthe three lots. No trends were present based on the number of sub-lotsprepared from values obtained on the initial lot consisting of 15sub-lots, the second lot that had 30 sub-lots and the third lotconsisting of 40 sub-lots. Assays have not been qualified for intra andinter variability therefore trend analysis and % CV comparison can onlybe made between starting and treated samples and the contribution ofassay variability and operator variability is presently not known. It isknown from manufacturer's literature that the presence of ozone andperoxide may give inaccurate values for the chlorine analysis. Also,redox analysis is pH dependent and the starting untreated saline mayrequire adjustment to pH 7 in order to determine if increases in redoxpotential are due to treatment or are just related to the differences inthe pH of the two products tested at the same time.

Osmolarity is in agreement with the calculated values that should beobtained based on the manufacturer's specification for percentage ofSodium Chloride present. (The freezing point depression at Δ° C. for a0.89% solution is 0.53. Osmolarity=Δ/1.86 or 0.285 Osm (285 mOsm). Thesevalues do not appear to change between non-treated, treated, nor overtime, or after stress treatment of freezing or boiling (Table 7).

TABLE 7 O time RT stored (Jul. 23, material Test 2004) Aug. 5, Ctrfreeze Ctr Boiled Performed treated 2004 Ctr thawed −20° c. 100 C. 1 minPH 6.99 7.1 7.0 6.52 Osmolarity 285 287 290 296 mOsm Peroxide 10 10 1010 Ppm Chlorine 72 52 77 32 Total mg/L (ppm) Chlorine 67 16 36 8 Freemg/L (ppm) Redox 830 830 840 870 mV Chloride 4670 5180 5260 4680 mg/L(ppm) Sodium 2470 2100 2000 2040 mg/L (ppm) Ozone 0.61 0.43 0.23 0.20mg/L (ppm)

Peroxide appears to increase and this increase appears to be stable tostress treatment. Ozone also increased post treatment but unlikeperoxide, appears to decrease over time and appears to be effected bystress treatments.

Levels of sodium and chloride in non-treated solutions are in agreementwith calculated values. Chloride post treatment appears to be withinassay error and appears to remain stable to stress treatment. Sodiumappears to decrease when starting concentration is compared to treatedsamples. The overall net decrease for the three runs gave a mean of1247+/−227 and appears to be statistically significant from assayvariability. These decreased values, however, do not appear to changewhen samples were stressed.

Levels of free and total chlorine and calculated combined chlorine maynot be valid due to interference from the presence of ozone andperoxide. Untreated starting material appears to have little if anymeasurable levels of chlorine. Post treatment values increase to a meanof 60 ppm for free and total indicating no combine chlorine is present.These values, however, might be influence by the presence of ozone andperoxide. It should also be noted that chlorine has a tendency to beabsorbed by plastics and may also be affected by the materials beingused to collect and store the sublets and final bulk materials as wellas the container used for sampling. Material stored for two weeks showeda change in the ratio of free and total and if calculated gave a valueof 36 ppm of combine chlorine with the values for ozone and peroxidebeing equivalent to the zero time treated test results that showedvalues of 60 ppm for both free and total indicating no combined chlorinepresent. If should also be noted that when stressed treated by heat, theozone values decreased and the total and free values for chlorine alsodecreased. The stressed treated samples at initial testing gave a valueof zero for combined chlorine, 36 ppm at 2 weeks and this sample whenboiled gave values of 24 ppm for combined chlorine and 41 ppm afterfreeze thaw.

Determination of Stress Effects of Temperature: Engineering run threewas stored at room temperature for two weeks in a PETG bottle. Thismaterial was re-tested after this period. Comparison of post treatmentmaterial from the 40 L pooled engineering Run #3 was originallyperformed and tested on Jul. 23, 2004. This material was stored at roomtemperature and samples taken and treated by freeze thawing and boilingto determine possible stability indicating assays. Data is shown inTable 2.

Sample preparation: Room Temperature sample removed directly fromoriginal container. Frozen sample was aliquoted into 50 ml (3×25 ml)conical tubes and frozen overnight. Sample was removed the followingday, brought to room temperature and tested.

Boiled sample: 75 ml was placed in a 125 ml flask, covered with tin foiland placed into water bath. Temperature was brought to 1000 C. Samplewas boiled for 1 minute and aliquoted into 50 ml conical tubes (3×25ml).

Conclusions: Additional testing will be conducted on the PQ runs todetermine reproducibility of the values obtain. Stability studies willalso be conducted to determine if variations are occurring over timewhen product is stored at refrigerated, room or elevated temperatures.Other testing by outside sources for biological activity is not yetavailable, however, storage containers, and time of holding may beimportant in determination of activity. Other testing for metals andleachable will be done as well as endotoxin and sterility on the PQpooled filtered samples.

TABLE 8 Table 8; Summary Data of Engineering Runs PretreatmentPost-treatment Parameter Mean 5.65 7.05 pH SD 0.84 0.04 Pre % CV 14.87 0.54 4.5-7.0 Range 4.39-6.91 6.99-7.10 Mean 284.67  285    Osmolarity SD0.44 0.00 mOsm exp % CV 0.16 0.00 277-326 Range  284-285.3   285-285.00Mean 0.00 10.00  Peroxide SD 0.00 0.00 ppm % CV 0.00 0.00 Range0.00-0.00   10-10.00 Mean 0.02 60.00  Chlorine SD 0.02 8.00 total % CV76.19  13.33  mg/L Range 0.00-0.05 48.00-72.00 Mean 0.01 59.33  Free SD0.00 5.11 mg/L % CV 33.33  8.61 Range 0.007-0.0  51.67-67.00 Mean320.50  860.53  Redox SD 67.67  20.36  mV % CV 21.11  2.37 Range 219-422.0   830-891.07 Mean 5140.00   4776.67   Chloride, SD 213.33 395.56  ppm exp % CV 4.15 8.28 5187-5509 Range  4820-5460.04183.333-5370.00  Mean 4140.00   3296.67   Sodium, SD 580.00  551.11 ppm exp % CV 14.01  16.72  3360-3571 Range  3270-5010.0   2470-4123.33Mean 0.01 0.49 Ozone, SD 0.01 0.12 ppm ppm % CV 66.67  24.20  (mg/L)Range 0-0 0.31-0.66

Example 14a

A composition comprising at least one redox signaling agent (RXN10) and50% NaOH were combined by first combining 75 μL 50% NaOH with 40 ml ofRXN10. Chlorine levels of this combination were measured to be 24 ppm.To this combination of RXN10 and NaOH, Carbopol® was added to make a 0.9wt % Carbopol®/99.1 wt % RXN10 mixture. The final chlorine levels ofthis combination were measured to be 9.6 ppm.

Example 14b

A composition made according to Example 1 (RXN9) and 50% NaOH werecombined by first combining 50 μL 50% NaOH with 40 ml of RXN9. Chlorinelevels of this combination were measured to be 22.6 ppm. To thiscombination of RXN9 and NaOH, Carbopol® was added to make a 0.9 wt %Carbopol®/99.1 wt % RXN9 mixture. The final chlorine levels of thiscombination were measured to be 9.2 ppm.

Example 14c

A comparison example was performed by combining 40 ml RXN10 and 0.36 gCarbopol® (0.9 wt % Carbopol®/99.1 wt % RXN10). The chlorine species forthis mixture was initially measured at 13.6 ppm. To this RXN10-Carbopol®mixture, 75 μL 50% NaOH was added which allowed for gelling. After a fewminutes, the chlorine species was measured at 7.6 ppm. After 48 hrschlorine was measured to be 1.6 ppm.

Example 14d

A second comparison sample was made with RXN9 and Carbopol® (1%Carbopol®=1.0 wt % Carbopol®/99.0 wt % RXN9). To this 1% Carbopol®/RXN9mixture, 263 μL of 50% NaOH was added. Chlorine in the final mixture wasmeasured to be 6.4 ppm. 48 h later the chlorine was 0.8 ppm and the pHwas 9.5.

Example 14e

A sample was prepared with RXN1O and Carbopol® (1% Carbopol®=1.0 wt %Carbopol®/99.0 wt % RXN1). To this 1% Carbopol®/RXN10 mixture, 225 μL of50% NaOH was added. To this 1% Carbopol®/RXN10/225 μL of 50% NaOHmixture, 100 μL of 12.5% NaOCl was added and the chlorine level wasfound to be 1 ppm.

Example 14f

Another sample was prepared with RXN9 and Carbopol® (1% Carbopol®=1.0 wt% Carbopol®/99.0 wt % RXN9). To this 1% Carbopol®/RXN9 mixture, 225 μLof 50% NaOH was added. To this 1% Carbopol®/RXN9/225 μL of 50% NaOHmixture, 50 μL of 12.5% NaOCl was added and the chlorine level was foundto be 1 ppm.

Example 14g

A third sample was prepared with RXN9 and Carbopol® (1% Carbopol®=1.0 wt% Carbopol®/99.0 wt % RXN9). To this 1% Carbopol®/RXN9 mixture, 277 μLof 50% NaOH was added. To this 1% Carbopol®/RXN9/277 μL of 50% NaOHmixture, 50 μL of 12.5% NaOCl was added and the chlorine level was foundto be 10.8 ppm.

Example 14h

Comparison samples were made with a 2 wt % Carbopol®/RXN9 mixture. Asample was prepared with RXN9 and Carbopol® (2% Carbopol®=2.0 wt %Carbopol®/98 wt % RXN9). To this 2% Carbopol®/RXN9 mixture, 500 μL of50% NaOH was added. To this 2% Carbopol®/RXN9/500 μL of 50% NaOHmixture, 50 μL of 12.5% NaOCl was added and the chlorine level was foundto be 27 ppm.

Example 14i

Another comparison sample was prepared with RXN9 and Carbopol® (2%Carbopol®=2.0 wt % Carbopol®/98 wt % RXN9). To this 2% Carbopol®/RXN9mixture, 500 μL of 50% NaOH was added. To this 2% Carbopol®/RXN9/500 μLof 50% NaOH mixture, 5 μL of 12.5% NaOCl was added and the chlorinelevel was found to be about 0 ppm.

Example 14j

To a 1% Carbopol®/RXN9 mixture, 225 μL of 50% NaOH was added asneutralizer, the pH was found to be neutral and the chlorine was 6 ppm.Subsequently, 100 μL of 12.5% OCl was added (pH of this mixture was 7)and the chlorine was immediately measured to be 21.2 ppm. After 5 min,the chlorine was measured to be 50 ppm. After an additional 5 min, thechlorine was measured at 52 ppm.

Example 14k

To a second batch of 1% Carbopol®/RXN9 mixture, 225 μL of 50% NaOH and100 μL of OCl were added, the pH was found to be 6 and the chlorine wasmeasured at 50 ppm.

Example 14l

A separate mixture of 1% Carbopol®/RXN9 was prepared, the pH was 3 andthe chlorine was measured as 13.2 ppm. To this 1% Carbopol®/RXN9mixture, 225 μL of 50% NaOH was added and the resulting combination hada pH of 6 and chlorine levels of 8.4 ppm. To this 1% Carbopol®/RXN9/225μL of 50% NaOH mixture, 50 μL of 12.5% NaOCl was added and the chlorinelevel was found to be 18 ppm.

Examples 14m-14n

Additional comparison examples were made comprising a first mixture of1% Carbopol®/RXN9, 225 μL of 50% NaOH and 50 μL of 12.5% NaOCl and asecond mixture of 1% Carbopol®/RXN9, 225 μL of 50% NaOH and 100 μL of12.5% NaOCl. The chlorine in the first mixture was measured as 19.4 ppmand the chlorine of the second mixture was measured as 54 ppm.

Examples 14o-14p

Another set of comparison examples were made comprising a first mixtureof 1% Carbopol®/RXN9, 225 μL of 50% NaOH and 50 μL of 12.5% NaOCl and asecond mixture of 1% Carbopol®/RXN9, 225 μL of 50% NaOH and 100 μL of12.5% NaOCl. The chlorine in the first mixture was measured as 30 ppmand the chlorine of the second mixture was measured as 53 ppm.

Example 15

pH was tested while increasing NaOH added to a 0.9% gel (0.9 wt %Carbopol®/99.1 wt % RXN9). Initial pH of a mixture of 0.9 wt % Carbopol®and 99.1 wt % RXN9 was 3.1. 50% NaOH was added incrementally as shownbelow.

Amount of 50% NaOH added pH Chlorine 100 μL  5.1 25 μL 6.0 25 μL 6.0 50μL 6.0 50 μL 9 3.8 ppm

Examples 16a-16b

Samples were made for APF testing comprising a first sample mixture of1% Carbopol®/RXN9, 225 μL, of 50% NaOH and 50 μL of 12.5% NaOCl and asecond sample mixture of 1% Carbopol®/RXN9, 225 μL of 50% NaOH and 100μL of 12.5% NaOCl. The chlorine in the first mixture was measured as26.4 ppm and the chlorine of the second mixture was measured as 36 ppm.APF values of the first sample mixture as 200% and the APF value of thesecond sample mixture was 136%.

Examples 16c-16d

Additional samples were made and tested for pH, chlorine levels, APF andRPE. A first sample mixture of 1% Carbopol®/RXN9, 250 μL of 50% NaOH and0 μL of 12.5% NaOCl was made and a second sample mixture of 1%Carbopol®/RXN9, 250 μL of 50% NaOH and 50 μL of 12.5% NaOCl was made.The results of each are shown in the table below:

First Sample Second Sample pH 6.5 pH 8 Chlorine 6 ppm Chlorine 30 ppmAPF 14% APF 200% RPE  0% RPE  26%

Examples 17a-17h

A composition comprising at least one redox signaling agent (RXN10) anda metal silicate are combined. Chlorine, APF and R-PE levels aremeasured.

A composition made according to Example 1 (RXN9) and a metal silicateare combined. Chlorine, APF and R-PE levels are measured.

A composition made according to Example 1 (RXN9) and a metal silicatehaving the following composition: SiO₂: 59.5%, MgO: 27.5%, Li₂O: 0.8%,and Na₂O: 2.8% are combined. Chlorine, APF and R-PE levels are measured.

A composition made according to Example 1 (RXN9) and a metal silicatehaving the following composition: SiO₂: 59.5%, MgO: 27.5%, Li₂O: 0.8%,and Na₂O: 2.8% are combined. The metal silicate is present at a weightpercentage of 2%. Chlorine, APF and R-PE levels are measured.

A composition made according to Example 1 (RXN9) and a metal silicatehaving the following composition: SiO₂: 59.5%, MgO: 27.5%, Li₂O: 0.8%,and Na₂O: 2.8% are combined. The metal silicate is present at a weightpercentage of 3%. Chlorine, APF and R-PE levels are measured.

A composition made according to Example 1 (RXN9) and a metal silicatehaving the following composition: SiO₂: 59.5%, MgO: 27.5%, Li₂O: 0.8%,and Na₂O: 2.8% are combined. The metal silicate is present at a weightpercentage of 4%. Chlorine, APF and R-PE levels are measured.

A composition made according to Example 1 (RXN9) and a metal silicatehaving the following composition: SiO₂: 59.5%, MgO: 27.5%, Li₂O: 0.8%,and Na₂O: 2.8% are combined. The metal silicate is present at a weightpercentage of 5%. Chlorine, APF and R-PE levels are measured.

A composition made according to Example 1 (RXN9) and a metal silicatehaving the following composition: SiO₂: 59.5%, MgO: 27.5%, Li₂O: 0.8%,and Na₂O: 2.8% are combined. The metal silicate is present at a weightpercentage of 6%. Chlorine, APF and R-PE levels are measured.

The terms “a,” “an,” “the” and similar referents used in the context ofdescribing the invention (especially in the context of the followingclaims) are to be construed to cover both the singular and the plural,unless otherwise indicated herein or clearly contradicted by context.Recitation of ranges of values herein is merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g., “such as”) provided herein isintended merely to better illuminate the invention and does not pose alimitation on the scope of the invention otherwise claimed. No languagein the specification should be construed as indicating any non-claimedelement essential to the practice of the invention.

It is contemplated that numerical values, as well as other values thatare recited herein are modified by the term “about”, whether expresslystated or inherently derived by the discussion of the presentdisclosure. As used herein, the term “about” defines the numericalboundaries of the modified values so as to include, but not be limitedto, tolerances and values up to, and including the numerical value somodified. That is, numerical values can include the actual value that isexpressly stated, as well as other values that are, or can be, thedecimal, fractional, or other multiple of the actual value indicated,and/or described in the disclosure.

Groupings of alternative elements or embodiments of the inventiondisclosed herein are not to be construed as limitations. Each groupmember may be referred to and claimed individually or in any combinationwith other members of the group or other elements found herein. It isanticipated that one or more members of a group may be included in, ordeleted from, a group for reasons of convenience and/or patentability.When any such inclusion or deletion occurs, the specification is deemedto contain the group as modified thus fulfilling the written descriptionof all Markush groups used in the appended claims.

Certain embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention. Ofcourse, variations on these described embodiments will become apparentto those of ordinary skill in the art upon reading the foregoingdescription. The inventor expects skilled artisans to employ suchvariations as appropriate, and the inventors intend for the invention tobe practiced otherwise than specifically described herein. Accordingly,this invention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

In closing, it is to be understood that the embodiments of the inventiondisclosed herein are illustrative of the principles of the presentinvention. Other modifications that may be employed are within the scopeof the invention. Thus, by way of example, but not of limitation,alternative configurations of the present invention may be utilized inaccordance with the teachings herein. Accordingly, the present inventionis not limited to that precisely as shown and described.

1. A stable hydrogel formulation comprising: a reactive oxygen species(ROS); saline; and a rheology modifier.
 2. The stable hydrogelformulation of claim 1, wherein the ROS comprises superoxide,hypochlorite, hypochlorate, oxygen derivatives, hydrogen derivatives,hydrogen peroxide, or hydroxyl radicals.
 3. The stable hydrogelformulation of claim 1, wherein the rheology modifier comprises a metalsilicate gelling agent.
 4. The stable hydrogel formulation of claim 1,wherein the rheology modifier comprises SiO₂, MgO, Li₂O, Na₂O, orcombinations thereof.
 5. The stable hydrogel formulation of claim 1,wherein the rheology modifier comprises a cross-linked acrylic acidpolymer.
 6. The stable hydrogel formulation of claim 1, wherein theformulation has a pH between about 5 and about
 9. 7. The stable hydrogelformulation of claim 1, wherein the formulation is formulated fortopical administration to a user.
 8. A stable hydrogel formulationcomprising: sodium present at a concentration of about 1000 to about2500 ppm; chloride present at a concentration from about 1200 to about5300 ppm; hypochlorous acid present at a concentration of about 16 toabout 67 ppm; superoxide radical present at a concentration of about 94μM; hydroxyl radical present at a concentration of about 241 μM; and arheology modifier present in an amount of about 0.1% to about 10% byweight.
 9. The stable hydrogel formulation of claim 8, wherein thecomposition has a pH between about 6 and about
 9. 10. The stablehydrogel formulation of claim 8, wherein the sodium, chloride,hypochlorous acid, superoxide radical and hydroxyl radical are measuredless than one year after the composition was made.
 11. The formulationof claim 8, wherein the formulation is formulated for topicaladministration to a user.
 12. The formulation of claim 8, wherein theformulation has an electron paramagnetic resonance (EPR) spectrum asshown in FIG.
 13. 13. (canceled)
 14. The formulation of claim 8, whereinthe rheology modifier comprises a metal silicate gelling agent, SiO₂,MgO, Li₂O, Na₂O, a cross-linked acrylic acid polymer, poly(acrylicacid), or combinations thereof.
 15. The stable hydrogel formulation ofclaim 1, wherein the formulation is stable for at least one year. 16.The stable hydrogel formulation of claim 1, wherein the ROS is presentin an amount of greater than about 98% relative to an initialconcentration of ROS for at least one year.
 17. The stable hydrogelformulation of claim 1, wherein the rheology agent is present in anamount of about 0.1% to about 10% by weight.
 18. The stable hydrogelformulation of claim 1, wherein the saline comprises sodium chloridepresent in an amount of about 0.1% to about 1.0% by weight.
 19. Thestable hydrogel formulation of claim 1, further comprising a bufferingagent, wherein the buffering agent comprises sodium phosphate monobasic.20. The stable hydrogel formulation of claim 8, wherein the formulationis stable for at least one year.
 21. The stable hydrogel formulation ofclaim 8, wherein the ROS is present in an amount of greater than about98% relative to an initial concentration of ROS for at least one year.